river, georgia j. stuart carlton abstract

Identification of Larval Moxostoma (Catostomidae) from the Oconee

River, Georgia

by

J. Stuart Carlton

(Under the direction of Cecil A. Jennings)

Abstract

Robust redhorse, Moxostoma robustum, is a recently rediscovered, imperiled

species of sucker (Catostomidae) that inhabits several rivers in the Atlantic Slope

drainage and is subject to intense conservation efforts. Its spawning period

frequently overlaps that of a sympatric congener, the notchlip redhorse (M.

collapsum), making identifying the larvae of the species difficult. I measured various

morphometrics, meristics, and developmental characteristics on lab-reared larvae of

each species, fit a classification tree model to the data, and used the model to create

a key discriminating between the species. The model had a leave-one-out,

cross-validation expected error rate of 4.7%. The key formed from the model is

highly accurate for fishes from 10–20 mm total length: three independent verifiers

used the key to identify fishes with a 95% accuracy rate. This key is one of a few

that distinguishes between sympatric Moxostoma larvae and is the first to identify

larval robust redhorse.

Index words: Robust redhorse, Moxostoma robustum, Notchlip redhorse,Moxostoma collapsum, Taxonomic key, Oconee River,Catostomidae, Larval fishes, CATDAT, Classification trees,Robust Redhorse Conservation Committee


River, Georgia

by

J. Stuart Carlton

B.A., Tulane University, 2001

A Thesis Submitted to the Graduate Faculty

of The University of Georgia in Partial Fulfillment

of the

Requirements for the Degree

Master of Science

Athens, Georgia

2004

c© 2004

J. Stuart Carlton

All Rights Reserved


River, Georgia

by

J. Stuart Carlton

Approved:

Major Professor: Cecil A. Jennings

Committee: Byron J. Freeman

James T. Peterson

Electronic Version Approved:

Maureen Grasso

Dean of the Graduate School

The University of Georgia

December 2004

Acknowledgments

Funding for this project was provided by the Athens office of the US Fish and

Wildlife Service and Georgia Power. Thanks is due to the Georgia Coop Unit and

the Warnell School of Forest Resources at the University of Georgia for providing

facilities and, of course, my education. I’d also like to thank Dr. Hank Bart; without

his advice and encouragement I’d probably still be selling beer at sporting events.

I received good technical advice from Nancy Auer, Rebecca Cull, David

Higginbotham, Haile MacCurdy, Wayne Starnes, Darrel Snyder, Paul Vecsei, and

Richard Weyers. My crew of technicians, who did much of the difficult work for this

project, included Gene Crouch, Peter Dimmick, Tavis McLean, Diarra Mosely, Dave

Shepard, and Steve Zimpfer. Bob Wallus made many of the measurements used in

my research and provided the written narratives in Appendices C and D.

My advisory committee was particularly helpful because of my non-scientific

background. Dr. Bud Freeman gave me wonderful advice on how to be a scientist.

Dr. Jim Peterson helped me understand the philosophy lurking behind the statistics,

and provided valuable computer programming assistance. Dr. Cecil Jennings was my

committee chair, field hand, confidant, and mentor. He taught me the importance of

maintaining balance in life and was wise enough to let me make my own mistakes.

He also showed me how to straighten out a trailered boat using a tree trunk.

I’d like to thank my family for putting up with me. I owe my sense of humor to

my mom; without it I wouldn’t have finished this project. My dad is my fishing

buddy, and is kind enough to re-rig my pole while I fish with his. Thanks, Dad.

Finally, I’d like to thank Libby for inspiring me to be my best, comforting me when

I wasn’t, and teaching me the value of a good checklist.

iv

Table of Contents

Page

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Chapter

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Appendix

A Key for Identifying Larval Moxostoma in the Oconee River,

Georgia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

B Characters Measured for the Classification Tree . . . . . . 46

C Development of Young Robust Redhorse . . . . . . . . . . . 56

D Development of Young Notchlip Redhorse . . . . . . . . . . 63

E Morphometric and Descriptive Measurements . . . . . . . . 72

v

List of Figures

1.1 Length-frequency distribution for larval Moxostoma collected May–

November 1996 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.2 Length-frequency distribution for larval Moxostoma collected April–

October 1999 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3.1 Morphometrics measured on notchlip and robust redhorse . . . . . . . 17

4.1 Standard length in laboratory-reared larval notchlip and robust redhorse 21

4.2 Pre-anal length in laboratory-reared larval notchlip and robust redhorse 21

4.3 Pre-dorsal fin length in laboratory-reared larval notchlip and robust

redhorse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

4.4 Greatest body depth in laboratory-reared larval notchlip and robust

redhorse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

4.5 Head length in laboratory-reared larval notchlip and robust redhorse . 23

4.6 Eye diameter in laboratory-reared larval notchlip and robust redhorse 23

4.7 Classification tree for the identification of larval notchlip and robust

redhorse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

4.8 Continuation of the classification tree for the identification of larval

notchlip and robust redhorse . . . . . . . . . . . . . . . . . . . . . . . 26

vi

List of Tables

4.1 Descriptive and ontogenetic traits used in the classification tree model 24

4.2 Description of traits used in the classification tree model . . . . . . . 27

B.1 Descriptive and ontogenetic traits measured on notchlip and robust

redhorse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

B.2 Description of traits measured on larval robust notchlip and robust

redhorse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

E.1 Morphometric and descriptive character measurements made on larval

notchlip and robust redhorse and fit to a classification tree model . . 73

vii

Chapter 1

Introduction

Robust redhorse (Moxostoma robustum) is a poorly known imperiled species of

large, riverine sucker (Teleostei: Catostomidae). Originally described as

Ptychostomus robustus by Edward Drinker Cope in 1870 (Cope 1870), robust

redhorse are presumed to be endemic to Piedmont and upper Coastal Plain rivers

along the Atlantic Slope drainage from the Pee Dee River in North Carolina to the

Altamaha River system in middle Georgia (Evans 1994). Anthropomorphic changes

to these rivers in the late 19th and early 20th centuries likely limited the habitat

available to robust redhorse, thereby greatly reducing their overall abundance

(Evans 1994). Overfishing also probably contributed to the population decline: they

were a widely-sought and heavily-harvested food fish in the 19th century (Cope

1870). Cope’s specimens were lost while being relocated (Bryant et al. 1996), and

the scientific community’s knowledge of robust redhorse disappeared with them

(Evans 1994). Indeed, the specific name robustus was errantly given to another fish

(Jenkins and Burkhead 1993).

In 1980, a robust redhorse was caught in the Savannah River, Georgia and

misidentified as a regional variant of the river redhorse (M. carinatum) (Jenkins and

Burkhead 1993). In 1985, another robust redhorse was caught in the Pee Dee River

in South Carolina and was similarly misidentified (Jenkins and Burkhead 1993). In

1991, five more robust redhorse were collected from the Oconee River in Georgia

(Evans 1994). Taxonomists, including Dr. Hank Bart, Dr. Byron Freeman, and Dr.

Robert Jenkins (Bryant et al. 1996), were perplexed by these catches and, after

1

2

reviewing regional catostomid systematics, they determined that the fish they had

identified as previously as a variant of M. carinatum were actually robust redhorse,

resurfacing after a 100 year absence (Jenkins and Burkhead 1993). The species was

rechristened M. robustum to conform with modern phylogenetic theory (Jenkins and

Burkhead 1993), and scientists began working to determine the species’ modern

range and what steps could be taken to conserve the rare fish (Evans 1996).

In 1995, a diverse group of stakeholders, including state and federal resource

agencies, universities, and private industrial companies, formed the Robust Redhorse

Conservation Committee (RRCC) to oversee the conservation efforts of the newly

rediscovered fish (Evans 1996). The goal of the RRCC is to reestablish robust

redhorse in sustainable numbers throughout its historical range without resorting to

listing the species under the Federal Endangered Species Act (Evans 1996).

Members of the RRCC have undertaken diverse projects toward this goal, including

assessments of artificial propagation techniques (Barrett 1997; Higginbotham and

Jennings 1999), annual population assessments (e.g., Jennings et al. 2000), genetic

determination of population diversity (Wirgin 2002), telemetric tracking studies

(Cecil A. Jennings, Georgia Cooperative Fish and Wildlife Research Unit, personal

communication), and various stocking regimes (e.g., Freeman et al. 2002).

These projects have achieved measured success. RRCC-approved research and

field work have led to the discovery of wild populations of robust redhorse in the

Oconee and Ocmulgee rivers in Georgia, the Savannah River in Georgia and South

Carolina, and the Pee Dee River in North Carolina (DeMeo 2001). Additionally, the

RRCC has established small stocked populations in the Ocmulgee, Broad, and

Ogeechee rivers in Georgia (DeMeo 2001). The successes of the RRCC have been

many, but there are still many critical areas where the understanding of robust

redhorse is incomplete.

3

Population recruitment is one such area. Age-0 redhorse rarely have been found

during the annual population assessments (Jennings et al. 1996). Additionally,

RRCC members have collected few robust redhorse between the sizes of 15 mm and

400 mm total length and don’t know how many robust redhorse survive past this

length or what happens to them if they do (DeMeo 2001). These facts suggest that

population recruitment may be limited (Jennings et al. 1996).

Understanding the population dynamics and ecology of the larvae may aid in

discovering the plight of the would-be recruits. To this end, the RRCC has

undertaken a variety of laboratory-based larval studies, including

swimming-strength evaluations (Ruetz III 1997), a study of the effects of gravel

quality on larval survival (Dilts 1999), and testing larval survival in various water

flow regimes (Weyers 2000). There also have been annual field studies of larval

abundance (Jennings, personal communication), but the data have been difficult to

analyze, as robust redhorse larvae are very similar in appearance to the larvae of a

sympatric congener, the notchlip redhorse (Moxostoma collapsum) (Wirgin et al.

2004). During spawning seasons with normal amounts of rain, robust and notchlip

redhorse spawn 3–6 weeks apart (see “Biology and Spawning Behavior”, below), and

their larvae are easy to distinguish based on size at capture (Figure 1.1) (Jennings,

personal communication). However, during years of abnormally low rainfall, the

spawning period of robust and notchlip redhorse is compressed, and size at capture

is not an effective means of distinguishing between them (Figure 1.2) (Jennings,

personal communication). One must use other methods.

A highly accurate alternative means of identification is to use unique genetic

identifiers within the fishes (Wirgin et al. 2004). However, this process is expensive

and time-consuming, (Jennings, personal communication). A taxonomic key

discriminating between the two species would facilitate the analysis of larval

abundance data by providing an inexpensive and quick method for identifying the

4

Figure 1.1: Length-frequency distribution for larval Moxostoma collected May–November 1996, a “normal” rain year. The larger clusters (above 35 mm total length)are assumed to be notchlip redhorse (M. collapsum) and the smallest one is assumedto be robust redhorse (M. robustum) (Jennings, unpublished data).

5

Figure 1.2: Length-frequency distribution for larval Moxostoma collected April–October 1999, a drought year. Notchlip redhorse (M. collapsum) and robust redhorse(M. robustum) cannot be identified by size at capture because of the large overlap inthe size classes (Jennings, unpublished data).

6

species, ideally while limiting the loss of accuracy compared to using genetic

identification. The goal of this project is to create such a key.

Biology and Spawning Behavior

Robust redhorse are typical members of genus Moxostoma: large, riverine,

bottom-feeding, and generally invertivorous, with an inferior mouth and thick,

fleshy lips (Jenkins and Burkhead 1993). One of the larger members of the genus,

robust redhorse can grow to 760 mm and reach 8 kg (Walsh et al. 1998). They are

among the most long-lived of the Moxostoma, often surpassing 20 years of age

(Evans 1994), compared to the 8–15 years of other redhorse (Jenkins and Burkhead

1993). Robust redhorse gather in the spring near shoals and flats to spawn over

coarse gravel substrate (Jennings et al. 1996). They spawn in groups of one female

and two or three males, quivering in unison to stir up the gravel as they release

their gametes (Jennings et al. 1996). Shuffling the gravel allows them to deposit

their eggs in the interstices at depths approaching 15 cm (Jennings et al. 1996). In

Georgia, spawning usually occurs from late April to early June, when water

temperatures reach approximately 19–20 ◦C (Jennings et al. 1996).

Notchlip redhorse are similar in appearance to robust redhorse. However, they

are slightly smaller when fully grown, with a maximum size of approximately 700

mm and 5 kg (Weyers 2000). Notchlip redhorse spawn in a similar manner to their

sister species (Jenkins and Burkhead 1993), gathering when the water temperatures

rise above 10 ◦C (Weyers 2000). In Georgia, this can occur anywhere from

mid-March to late April (Weyers 2000).

Chapter 2

Literature Review

Proper species identification is an essential part of ecological research. Identifying

fish requires a consistent protocol to ensure accuracy and precision. Identifying

fishes often is a difficult task, and poorly-explained or improperly followed protocols

can render it impossible.

Identification of Adult Fishes

The basic methodology for identifying adult fishes has been in place for over a

century. Edward Drinker Cope, who described over 300 new species of fish between

1862 and 1894 (Academy of Natural Sciences 2004), devised many of the

characteristics that are used to identify adult fishes today. However, the

characteristics weren’t standardized, and taxonomists’ work was subjective and

difficult to repeat (Hubbs and Lagler 1958). Subjectivity was the rule until 1958,

when Carl L. Hubbs and Karl F. Lagler published Fishes of the Great Lakes Region,

which contained the the first widely-available attempt to standardize the definitions

of the most commonly used taxonomic characters (Hubbs and Lagler 1958).

Taxonomic characters are divided into two main categories: meristics and

morphometrics. Meristics, which are generally the more reliable of the two (Fuiman

1979) are aspects of a fish that can be counted (Hubbs and Lagler 1958). Commonly

useful external meristics for identifying adult fishes include number of scales along

7

8

the lateral line; number of pre-dorsal scales; number of circumpeduncal scales; and

dorsal, anal, caudal, pectoral, and pelvic fin ray counts (Strauss and Bond 1990).

Commonly useful internal meristics include number of gill rakers, amount of various

types of dentition, and number of vertebrae (Strauss and Bond 1990). Meristic traits

are useful because they usually are easy to count, but they can be influenced by

environmental factors, especially temperature (Lindsey 1958; Lindsey 1962; Barlow

1961).

Morphometrics are body measurements and proportions (Hubbs and Lagler

1958). The most common include head length, snout length, eye orbit length, body

depth, pre-anal length, length of the longest dorsal fin ray, and the heights of

various fins. These measures usually are expressed as a percentage of the standard

length or total length of the specimen to remove the effects of the size of the fish

(Strauss and Bond 1990). Morphometrics can be affected by environmental factors

— particularly diet — throughout the life of the fish, which can limit their

diagnostic utility (Snyder and Muth 1990; Van Velzen et al. 1998).

In addition to meristics and morphometrics, other anatomical characters, such

as pigmentation, descriptions of lateral line shape, position, and completeness, and

secondary sexual characteristics, such as the presence or absence of breeding

tubercles, often are used on a case-by-case basis (Strauss and Bond 1990). The

appearance of unusual characters often provides conclusive evidence to a difficult

taxonomic problem. As with meristics and morphometrics, anatomical traits —

especially pigmentation (Bolker and Hill 2000) — can be affected by environmental

conditions, so they must be used judiciously (Snyder and Muth 1990).

Identification of Larval Fishes

The technique for identifying larval fishes is similar to that for identifying adult

fishes. However, many of the adult characters are not present or are less-developed

9

in larval fishes, and are ineffective for discriminating between species (Snyder and

Muth 1990). A taxonomist often must use modified versions of adult characters or

unique larval characters to discriminate among larval fishes (Methven and

McGowan 1998). The effective — and present — characters vary among families

and even among genera (Snyder and Muth 1990). Additionally, larval fish characters

vary greatly with the age and size of a fish (Kendall et al. 1984), so taxonomists

must study developmental series of larvae and young juveniles at different sizes and

often must treat size classes or developmental stages as entities distinct from each

other (e.g., Fuiman 1979, Snyder 1983, Snyder and Muth 1990, Wallus et al. 1990).

In practice, larval identification papers tend to be one of several types:

traditional dichotomous keys (e.g., Fuiman 1982, Snyder and Muth 1990, and Kay

et al. 1994), descriptions of diagnostic traits without a dichotomous key (e.g.,

Karjalainen et al. 1992 and Snyder 2002), or comparisons of obtained samples to

previously-published descriptions (e.g., Bunt and Cooke 2004). The utility of these

formats varies.

Comparisons of samples to previously-published literature are relatively easy to

make because they only require obtaining larvae of the new species. However, there

are a number of disadvantages to this technique: the published description may be

inadequate for species discrimination (e.g., Fuiman and Witman 1979; Moxostoma

in Kay et al. 1994); statistical analysis is difficult or impossible without access to

the data from the prior description; and the comparisons often are made for fishes

from different geographical regions (e.g., Bunt and Cooke 2004, which distinguishes

Moxostoma valenciennesi from other catostomids based on descriptions of fishes in

Tennessee published in Kay et al. 1994). Given the inherent potential for

environmentally induced variability in meristic, morphometric, and pigmentation

patterns (see “Identification of Adult Fishes”, above), diagnostic characters for a

species in one region may not remain consistent throughout all regions.

10

A simple description of diagnostic traits is sufficient when there is a character

that consistently distinguishes between the species (e.g., Snyder 2002). However, if

there are several species to be identified, or the diagnostics characters change based

on fish size or are interrelated, then a dichotomous key, which is a more flexible

presentation, is appropriate.

Modern Identification Techniques

In addition to the traditional methods, there have been several recent advances in

identifying fishes. Landmark-based morphometric (LBM) analysis involves using

computers and video-capturing software to analyze body proportions based on

morphological landmarks on the body (Rohlf and Marcus 1993; Edwards and Morse

1995; Fulford and Rutherford 2000). Although this method can be accurate (Fulford

and Rutherford 2000), the technology required for LBM is not widespread and isn’t

as useful as a traditional key.

Another new identification technique involves analyzing various genetic traits to

identify species (Lindstrom 1999; Tringali et al. 1999; Wirgin et al. 2004). Genetic

analysis has is highly accurate, but requires specialized training and expensive

equipment (Jennings, personal communication). Another promising use of genetic

analysis is to test the accuracy of previously-made keys (Wirgin et al. 2004). This

gives a taxonomist a good idea of the accuracy of a key while incurring only a

one-time cost.

Both LBM and genetic analysis are theoretically superior to traditional

key-based identification. In the future, larval identification will largely comprise

these techniques. In the interim, key-based identification remains the simplest,

cheapest, and most widely-available technique for distinguishing between larval

fishes.

11

Statistical Analysis in Larval Keys

Statistical analysis in larval identification has been inconsistent. Keys often are

published without any discussion of statistics (e.g., Wallus et al. 1990, Kay et al.

1994, Urho 1996, Snyder 2002). When there is a statistical analysis described (e.g.,

Fuiman 1979) there is rarely a “real world” test of the key, so it isn’t clear how

accurately lay users can identify fishes with the key. Although differences between

species can seem drastic enough not to require a thorough statistical analysis, keys

published without any statistical verification are difficult to assess from afar.

The primary statistical tools used for analyzing meristic and morphometric data

include Student’s t-tests (Urho 1996), analysis of variance (ANOVA), and principal

components analysis (PCA) (Libosvarsky and Kux 1982; Mayden and Kuhajda

1996; Van Velzen et al. 1998). ANOVA often is performed using arcsine-transformed

data to remove the effects of size (Sokal and Rohlf 1981). This is a somewhat

controversial procedure (Atchley et al. 1976; Packard and Boardman 1988; Prairie

and Bird 1989; Jackson and Somers 1991), as biologists tend to misinterpret or

overstate the value of such transformations. When other techniques fail, PCA can

used to summarize covariation by using newly formed characters (called principal

components) (Jolliffe 1986). Mayden and Kuhadja (1996) also used sheared PCA to

remove the effects of fish size.

There are other methods for analyzing the meristic and morphometric data.

Mayden and Kuhadja (1996) also used analysis of covariance on untransformed

morphometric data. Discriminant function analysis (DFA) can be used when there

is a high morphometric and meristic similarity between species (Fuiman 1979;

Libosvarsky and Kux 1982; Methven and McGowan 1998). DFA combines the

discriminating value of several characters to determine whether one group of

characters is significantly different from another (Libosvarsky and Kux 1982), which

is useful when a single character does not distinguish between the species

12

(McAllister et al. 1978). Like ANOVA, DFA requires either normally distributed

data (Methven and McGowan 1998) or data transformed to approximate a normal

distribution (Lachenbruch 1975; Pimentel 1979; Harris 1985).

There is a little-used technique called tree-based classification that classifies

categorical responses without requiring any specific distribution (Breiman et al.

1984). Classification trees are created through recursive partitioning: dividing data

into increasingly homogenous subsets (based on a set of response variables) until a

specified degree of homogeneity is achieved (Breiman et al. 1984). Each division is

called a node, and once the partitioning is complete, each terminal node is the

model’s predicted response (Breiman et al. 1984). Tree-based classification is

particularly well-suited for creating taxonomic identification keys because it is

flexible enough to analyze combinations of quantitative and qualitative data

(Breiman et al. 1984) such as morphometric measurements and pigmentation

pattern descriptions (Weigel et al. 2002).

Catostomid Research

Taxonomic keys for larval catostomids—particularly Moxostoma—are scarce. Of the

few available, the ones most relevant to this project are studies of catostomids done

by Fuiman (1979), Fuiman and Witman (1979), Fuiman (1982), Snyder and Muth

(1990), Kay et al. (1994), and Bunt and Cooke (2004). Fuiman (1979) described and

identified several larval catostomids, including shorthead redhorse (M.

macrolepidotum) from Northern Atlantic Slope drainages. Fuiman and Witman

(1979) unsuccessfully attempted to distinguish between shorthead redhorse and

golden redhorse (M. erythrurum) from the same region. Fuiman (1982) was finally

successful in distinguishing between the species in what may be the only published

English-language key to distinguish between sympatric Moxostoma in North

13

America without relying on previously-published descriptions (e.g., Bunt and Cooke

2004).

Snyder and Muth’s (1990) thorough study described and distinguished between

the larvae of several catostomid species in the upper Colorado River system. Their

key represents a high-water mark in terms of detail and complexity. With

approximately 1000 couplets, it illustrates how to identify exceptionally similar fish

by using extreme specificity. Kay et al. (1994) described catostomids in the Ohio

River drainage, including golden redhorse, shorthead redhorse, silver redhorse

(Moxostoma anisurum), river redhorse (M. carinatum), and black redhorse (M.

duquesnei), but were unable to satisfactorily distinguish among them.

Despite the small literature base, there seems to be a growing interest in

catostomids. There have been several comprehensive reviews of catostomid

systematics published in recent years (Bunt and Cooke 2004). This study will join

what hopefully will be a growing base of knowledge about the family.

Chapter 3

Methods

Specimen Collection

Notchlip redhorse broodstock were collected by using boat electrofishers along

several sites on the Oconee and Broad rivers in middle Georgia during the spawning

season of 2003. If a male-female couple could be found, any fish running ripe were

strip-spawned in the field. Field-fertilized eggs were submerged in approximately 15

cm of river water in a small (≈12 L) cooler. The water in the cooler was aerated

with a small, battery-operated aerator. Fish that were not running ripe were taken

in holding tanks to the University of Georgia Whitehall Fisheries Research Lab in

Athens, Georgia for hormonally-induced spawning.

To artificially induce spawning, the notchlip redhorse were injected with

OvaprimTM, a liquid peptide supplement that effectively induces spawning in

Moxostoma robustum (Barrett 1997). The total dose of OvaprimTMgiven to females

was 0.5 mL per kg of body weight. The total dose given to males was 0.05 mL per

kg of body weight. Since the gender of the fish wasn’t known, all fish were given an

initial “priming” dose of 0.05 mL/kg, which was approximately a total dose for a

male. Fish that didn’t respond after twelve hours were assumed to be female and

were given a 0.45 mL/kg resolving dose. After the OvaprimTMtreatment, the fish

were checked every 12 hours for milt or egg production. When a ripe male-female

couple was found, the fish were strip-spawned and the eggs were fertilized manually.

Fertilized eggs collected in the field and in the lab were placed in 37-L aquaria at

a density of approximately 300–500 eggs per aquarium. The bottom of each

14

15

aquarium was lined with eight to 10 small (≈60 mm diameter) rocks to provide

shelter for newly-hatched larvae. A combination of ambient and florescent light was

used to keep the aquaria on a light cycle consistent with the solar cycle at the time.

Water in the aquaria was kept at ambient temperature, ranging from 18–22 ◦C. The

water was changed twice per day for the first week after the eggs were fertilized and

daily in subsequent weeks.

After hatching and the development of mouth parts, the larvae were fed a

combination of commercial larvae feed, based on the suggestions made by

Higginbotham and Jennings (2000), and Artemia spp. Several larvae were sampled,

euthanized, and stored in 10% buffered formalin every 12 hours for the first week

after hatching and every 24 hours during subsequent weeks. Originally, six larvae

per day were sampled, but this number was later reduced to three to ensure that an

adequate number of larvae from each size class were sampled. The larvae were

stored for at least two months before data collection to allow time for shrinkage.

M. robustum used for data collection were obtained from a reference collection

at the Georgia Cooperative Fish and Wildlife Research Unit at the University of

Georgia in Athens, Georgia. The reference collection was a developmental series

reared in a laboratory from wild-caught parents of known identification. The

reference collection was stored in 10% formalin. Additionally, data taken from

different, laboratory-reared M. robustum for a previous study (Looney and Jennings

2005) were analyzed.

Measurements and Data Collection

A stereo dissecting microscope at 10x magnification was used for all measurements

on each sample. Jaw-type dial calipers or an ocular micrometer were used to

measure several morphometrics, including total length, standard length, pre-anal

length, pre-dorsal fin length (where appropriate), greatest body depth (on

16

post-yolk-sac larvae), head length, and eye diameter (Figure 3.1). Morphometrics

were measured by an expert larval taxonomist (Robert Wallus of Murphy, North

Carolina) to ensure that operator error did not contribute to the observed

differences between the two species. Morphometrics were defined as follows (based

on Wallus et al. 1990):

Total length: Straight-line distance from the anterior-most part of the head to

the tip of the tail or caudal fin.

Standard length: Straight-line distance from the anterior-most part of head to

the most posterior point of the notochrod or hypural complex.

Pre-anal length: Distance from the anterior-most part of the head to the

posterior margin of the anus.

Pre-dorsal fin length: Distance from the anterior-most part of the head to the

anterior margin of the dorsal fin. Measured in larvae with dorsal fin

development.

Head length: Distance from the anterior-most tip of the head to the

posterior-most part of the opercular membrane, excluding the spine; prior to

opercular development, measured to the posterior end of the auditory vesicle.

Eye diameter: Horizontal measurement of the iris of the eye.

Greatest body depth: Greatest vertical depth of the body excluding fins and

finfolds. Measured on post yolk-sac larvae.

All measurements were at least to the nearest 0.1 mm, and some to the nearest 0.05

mm.

The expert taxonomist also provided qualitative narrative descriptions of the

developmental progress of each species (Appendices C and D). These narratives

17

Figure 3.1: Morphometrics measured on Moxostoma robustum and M. collapsum (notto scale).

18

were used as the basis for quantitative analysis of descriptive traits. Descriptive

traits measured included pigmentation patterns (observed with a polarized light

filter) and total length of fish at the time of certain ontogenetic events (such as yolk

absorption, finfold development, and the development of fins) (Table B.2).

Descriptive characters were scored as either present, absent, or undeveloped and

used in the statistical analysis. Several meristics (including myomere and fin ray

counts) were measured, but were not used for the statistical analysis because they

were found previously to be non-diagnostic (Looney and Jennings 2005). The

notchlip redhorse larvae used for data collection were archived in the Georgia

Museum of Natural History (accession number GMNH4434) for future reference.

Statistical Analysis and Key Creation

Quantitative measurements were tested for differences between the species by

overlaying plots of their relationship to total length in each species (PROC GPLOT,

SAS Institute) and looking for divergence between the two species. Chi-square tests

of association were used to test for significant (α=0.05) differences in the categorical

measurements between species (Snedecor and Cochran 1989). 14 traits were selected

based on a combination of ease-of-use and statistical significance for further

analysis. CATDAT, a computer program for categorical data analysis (Peterson et

al. 1999), was used to fit a classification tree model to the data.

TL was included in the tree model to explicitly incorporate the morphological

development that occurs as the fish grows. This approach obviates the need to make

a separate tree and key for each millimeter size class. The classification tree was

kept to 14 other traits (either qualitative or quantitative) because of technical

limitations of the CATDAT program.

CATDAT is capable of generating many different tree models based on user

specification of several variables, including tree size and size of each “partition”, or

19

subset, of the data (Peterson et al. 1999). The final model was chosen to minimize

both tree size (number of nodes) and the expected error rate (EER) of the model.

The EER of the model was estimated using leave-one-out cross validation, which

has been found to be an almost unbiased estimator of EER (Fukunaga and Kessel

1971). The key was checked for accuracy and ease-of-use by 3 independent verifiers

using laboratory-reared larval robust and notchlip redhorse of known identity. The

broodstock used to produce these larvae were collected at a different time than

those that produced the larvae used to create the model. Each verifier tested the key

on two samples of 25 larvae of each species, for a total of two replicates of 50 fishes.

The verifiers had a variety of experience using larval keys: one with less than 1 year

experience working with larval keys, one with 5 years of experience, and one with 20

years of experience. Their suggestions on improving clarity and ease-of-use were

incorporated into the key after they all completed both replicates of the verification.

Chapter 4

Results

Notchlip Redhorse Broodstock Collection and Spawning Induction

Twenty-nine notchlip redhorse were collected from various sites in the Oconee and

Broad Rivers. Of these, two (one male and one female) from the Broad River were

running ripe and were strip-spawned in the field, yielding approximately 2000

fertilized eggs. The remaining 27 were taken to the University of Georgia Whitehall

Fisheries Research lab for artificially-induced spawning.

Twenty notchlip redhorse (16 females and four males) were treated with

OvaprimTMto induce gonadal production. Seven (four females and three males)

responded to the treatment and reached spawning condition. However, the response

of the males and females was asynchronous, and fertilized eggs were not obtained.

Morphometrics

Morphometrics were measured on 68 notchlip redhorse larvae (hatched from the

Broad River-collected eggs) and 101 robust redhorse (hatched from Oconee

River-collected eggs). The size of the larvae ranged from 9.0–21.0 mm TL for

notchlip redhorse and 7.2–22.7 mm TL for robust redhorse. Of the morphometrics

measured (Figure 3.1), only pre-anal length as a percent of total length showed

divergence between the two species throughout the size range (Figures 4.1–4.6) and

was used in the classification tree. The remaining morphometrics showed either

little divergence or only diverged over a part of the size range of the larvae. These

characteristics were omitted from the classification tree model.

20

21

Figure 4.1: Standard length (expressed as % total length) in laboratory-reared larvalnotchlip redhorse (Moxostoma collapsum) and robust redhorse (M. robustum).

Figure 4.2: Pre-anal length (expressed as % total length) in laboratory-reared larvalnotchlip redhorse (Moxostoma collapsum) and robust redhorse (M. robustum).

22

Figure 4.3: Pre-dorsal fin length (expressed as % total length) in laboratory-rearedlarval notchlip redhorse (Moxostoma collapsum) and robust redhorse (M. robustum).

Figure 4.4: Greatest body depth (expressed as % total length) in laboratory-rearedlarval notchlip redhorse (Moxostoma collapsum) and robust redhorse (M. robustum).

23

Figure 4.5: Head length (expressed as % total length) in laboratory-reared larvalnotchlip redhorse (Moxostoma collapsum) and robust redhorse (M. robustum).

Figure 4.6: Eye diameter (expressed as % total length) in laboratory-reared larvalnotchlip redhorse (Moxostoma collapsum) and robust redhorse (M. robustum).

24

Table 4.1: Descriptive and ontogenetic traits used in the classification tree analysis.The size-class(es) for which the trait is significantly distinctive between the species islisted. A more detailed description of the traits appears in Table 4.2.

Character measured Size class (mm) p-value of χ2

Head position 10 < 0.001Head position 11 < 0.001Notochord flexion 10 < 0.001Notochord flexion 11 < 0.001Eye pigment 11 0.001Eye pigment 12 < 0.001Myosepta pigment 11 < 0.001Digestive tract 13 < 0.001Dorsal fin 13 < 0.001Yolk sac 14 < 0.001Dorsal fin margins 14 < 0.001Anal fin 14 < 0.001Anal fin 15 < 0.001Pelvic flaps 15 0.001Lip pigment 16 0.003Snout pigment 17-20 0.003

Descriptive Characters

Fifty-nine descriptive and ontogenetic traits were scored on 149 of the fishes (68

notchlip redhorse from the Broad River and 81 robust redhorse from the Oconee

River). Of the traits measured, 13 were selected for inclusion in the classification

tree analysis, with at least one significant difference between the two species selected

from each millimeter size-class (Tables 4.1 and 4.2). Total length and pre-anal

length also were included in the classification tree analysis.

25

Classification Tree Model

The classification tree models were fit to measurements of 149 fishes. The final

model was selected to minimize both tree size and expected error rate. The

classification tree chosen was a 24-node tree with 12 terminal nodes and 12

non-terminal nodes (Figures 4.7–4.8. The leave-one-out cross-validation expected

error rate was 4.7%. The prediction error rate for notchlip redhorse (i.e., the number

of fishes classified by the model as notchlip redhorse that were actually robust

redhorse) was 0%. The prediction error rate for robust redhorse was 7.95%. The

classification tree model was used to form the key found in Appendix A.

Key Validation

The key was tested by three independent testers in two replicates of 50 fishes (25 of

each species). The overall average accuracy rate for the three testers over two

replications was 95%. Tester A, who had approximately 20 years of experience

identifying larval fishes, correctly identified 48 of 50 fishes (96%) in each replication.

Tester B, with approximately five years of experience, correctly identified 47 of 50

fishes (94%) in the first replication and 48 of 50 fishes (96%) in the second

replication. Tester C, who had less than one year of experience, correctly identified

47 of 50 fishes (94%) in each replication. In the first replication, six of the eight

errors (75%) were notchlip redhorse misidentified as robust redhorse. All six

notchlip redhorse errors were the result of two specimens that were misidentified by

all three testers. The source of the misidentification (i.e., couplet) was not consistent

among testers. Testers B and C each uniquely misidentified one robust redhorse as a

notchlip redhorse. Although the couplet leading to the robust redhorse

misidentification was not consistent, each error was the result of a total length

measurement that was incongruent with proper identification of the specimen.

26

Figure 4.7: Classification tree for the identification of larval robust redhorse (Moxos-toma robustum) and notchlip redhorse (M. collapsum). Descriptions of the predictorsappears in Table 4.2. The diagram continues in Figure 4.8

Figure 4.8: Continuation of the classification tree for the identification of larval robustredhorse (Moxostoma robustum) and notchlip redhorse (M. collapsum). Descriptionsof the predictors appears in Table 4.2. The diagram begins in Figure 4.7

27

Table 4.2: Descriptive and ontogenetic traits used in the classification tree model.Traits were measured on fishes of all sizes. Traits were scored as the more advancedstate for all remaining size classes once the trait became present in all specimens in agiven size class. In cases where the trait has a more and less advanced state, the lessadvanced state is listed first.Character DescriptionHead position Curved: head curved against yolk sac

Lifted: head lifted away from yolk sacNotochord flexion Straight: tip of notochord not flexed

Flexed: tip of notochord flexedEye pigment Unpigmented: middle of eye yellowish or unpigmented

Pigmented: middle of eye with brown or black pigmentMyosepta pigment Unpigmented: pigment absent on median myosepta

Pigmented: dashed line of pigment along median myoseptaDigestive tract Not functional: digestive tract development incomplete

Functional: digestive tract fully developed and functionalDorsal fin Absent: dorsal fin and dorsal fin profile absent

Developing: developing dorsal fin or dorsal fin profile evidentYolk sac Present: yolk sac present

Absent: yolk sac completely absorbedDorsal fin margins Undefined: margins of dorsal fin undefined

Defined: anterior and posterior margins fin well-definedAnal fin Absent: anal fin absent; development has not begun

Developing: anal fin development has begunPelvic flaps Absent: pelvic fin development has not begun

Flaps: pelvic flaps developingLip pigment Absent: pigment absent on upper lip

Upper: pigment present on upper lipSnout pigment Absent: melanophores absent across snout

Bar: bar of small melanophores present across snout

28

In the second replication, four of the seven identification errors (57.1%) were

notchlip redhorse misidentified as robust redhorse. Three of the notchlip redhorse

errors were the result of a single specimen misidentified by all three testers. All

three testers misidentified the specimen at couplet 8 of the key (digestive tract

development). The remaining notchlip redhorse identification error was from a

specimen uniquely misidentified by tester C at couplet 2 (total length). Two of the

robust redhorse identification errors were the result of a single specimen

misidentified as a notchlip redhorse by testers A and C. Each of these errors were

made at couplet 3 (total length). The remaining robust redhorse identification error

was a specimen uniquely misidentified by tester B at couplet 9 (eye pigment).

Chapter 5

Discussion

Key Development and Strategic Approach

The classification tree model yielded a key that is effective at discriminating

between larval robust and notchlip redhorse from hatch to 20 mm total length (TL).

To my knowledge, it is the first key to successfully distinguish between sympatric

early-stage larval Moxostoma in the southern United States. Indeed, there have

been few successful keys made for larval Moxostoma in any region. Fuiman (1982)

successfully distinguished between larval golden redhorse (M. erythrurum) and

shorthead redhorse (M. macrolepidotum) in the Great Lakes region after an earlier

failed attempt using fishes from the Great Lakes and northern Atlantic Slope

drainages (Fuiman and Witman 1979). There have been other attempts (e.g., Bunt

and Cooke 2004) to distinguish between larval Moxostoma by comparing published

descriptions with an on-hand collection, but there hasn’t been a statistically-verified

key. This is also the first key to identify larval robust redhorse: such a feat wasn’t

even possible until the recent rediscovery of the species.

Assessing this key’s accuracy relative to previously published keys is difficult,

because most published keys provide minimal description of statistical methods

used and lack discussion of identification error rate. Fuiman (1979) is an exception;

his key for five northern Atlantic Ocean drainage catostomid species accurately

identified 82.6 to 100% of the species, depending on developmental stage. However,

Fuiman’s key does not attempt to identify any congeners, which is a more difficult

29

30

task. In light of this, the 95% “real world” accuracy rate achieved with the

newly-developed key is satisfactory.

The couplets in the key presented here are almost exclusively based on TL at

the occurrence of certain ontogenetic events. Ontogenetic timing is effective because

newly-hatched robust redhorse are 1–2 mm smaller in TL than newly-hatched

notchlip redhorse and are more ontogenetically advanced at a similar size. For

example, an 11 mm TL robust redhorse may be several weeks old, whereas a

notchlip of the same length may only be several days old. The robust redhorse

would have had more time to feed and grow than the notchlip and would have

reached a more advanced life stage.

Ontogenetic timing often is used in larval keys, but usually only in a few

couplets (e.g., several of the keys in Hogue, Jr. et al. 1976 and Kay et al. 1994) or in

combination with other characters (e.g., Fuiman 1982). This key is unusual in that

it attempts to distinguish between sympatric congeners that are very similar in

appearance. Since I was unable to find any easily measured meristic, morphometric,

or pigmentary traits that differed consistently as the larvae grew, I relied on

ontogenetic timing to keep the key as simple and user-friendly as possible and to

avoid the difficulties of a very complex key. Kay et al. (1994) faced a similar

conundrum trying to identify the larvae of catostomids in the Ohio River drainage

and made a similar choice.

The downside of using ontogenetic timing is that the effect of environmental

variation (temperature, current, dissolved oxygen, food supply, light regime, etc.) on

the development of the fishes is unclear. I tried to minimize the effect of these

unknown factors by choosing several different types of developmental characters

(pigment, internal organ, and fin development). My goal was to eliminate as much

error as possible from the entire fish identification process, not just from the model

used to make the key.

31

The alternative to relying on ontogenetic timing is to either give up and only

identify fishes to family or genus (e.g., Moxostoma in Kay et al. 1990) or to use very

complex and difficult-to-measure characters. Snyder and Muth (1990) used the

latter approach to identify six catostomid species, and the resulting key is

exceptional in both its thoroughness and complexity. Snyder and Muth’s (1990) key

has approximately 1000 couplets, identifying the fishes from hatch through early

juvenile stages with a variety of meristics, morphometrics, pigment descriptions, and

ontogenetic traits. Although Snyder and Muth (1990) do not provide a statistical

analysis of their key, the model is presumably accurate if used properly by an expert

taxonomist. I don’t believe that the key is appropriate for lay users, and it’s

complexity may even lead to a higher error rate because of confusion or improper

measurement. My goal was to eliminate as much error as possible from the entire

fish identification process, not just from the model used to make the key.

Several of the predictors used to fit the model to the data were not included in

the final classification tree. These traits include pre-anal length, notochord flexion,

dorsal fin development, pelvic fin development, lip pigmentation, and snout

pigmentation. These probably were left out of the model because they were

autocorrelated with other characters and therefore not predictive.

Accuracy of Identification Using The Larval Key

There are three major sources of potential error in identifying larval fishes: the

accuracy of the model used to make the key, the precision of the key users, and

variables associated with the fishes being identified. I attempted to minimize the

overall error of the identification process by creating a key that would limit the

error in each individual component.

The classification tree model had an expected error rate of 4.7%, which

compares favorably to previously published keys (e.g., Fuiman 1979). The model as

32

constructed is based on a relatively small sample of limited genetic diversity;

nonetheless, tests of the key performed on fishes of a wider genetic background

showed the model to be accurate, and should limit concerns about sample size.

There should be minimal identification error based on the key itself.

Larval keys often are plagued by difficult-to-measure characters or complex

counts that are nearly impossible to make consistently. A highly accurate key is

useless if the users can’t properly measured the diagnostic traits. Therefore, I

included ease-of-measurement in my criteria for selecting traits to minimize error

associated with the users or the key. The similarity of the error rate of the key

verifiers (5%) compared to the expected error rate of the model (4.7%) suggests that

these attempts were successful. Including internal characters or other very

complicated traits in the model may have made it more accurate or adaptable to

environmental variation, but the cost of the increased accuracy would have been a

substantial decrease in the user-friendliness of the key. I wanted to avoid creating a

key as complex as Snyder and Muth’s (1990), and was unwilling to trade a slightly

more accurate model for a much more difficult-to-use key.

Limiting error associated with the model and the key users should minimize the

impact of uncontrollable variables, such as the condition of the fishes being

identified, on the identification process. Larval fishes are fragile and difficult to

collect; those caught in the field may be in poor physical condition because of

damage that occurred during sampling. They also will have developed under

different environmental conditions than the larvae used to form the key. These

factors could lead to misidentification. The key has at least minimal plasticity,

however: the second-party verifiers successfully identified fishes raised in the lab

under differing sets of conditions. Any remaining questions about the keys accuracy

on wild-caught fishes will be cleared up in the near future, when wild-caught,

genetically-identified fishes will be used to test the key.

33

Limitations of the Key

The most important limitation of the key is the reliance on total length. Improper

measurement of TL may cause misidentification of the specimen in question.

Additionally, specimens that have immeasurable TL because of damage or deformity

cannot be reliably identified with this key.

Another important limitation is that the data used to make the key were

collected entirely from fishes preserved in 10% buffered formalin. Other

preservatives, such as ethanol, may cause differential shrinkage and invalidate the

key. A separate key may need to be developed for such fishes.

The scarcity of larvae of both species limited the scope of the key, which is

accurate only to approximately 20 mm TL. I decided to allocate the larvae to make

a key that was accurate over a smaller range rather than one that was less accurate

over a larger range.

The goal of the project was to create a key that would identify the fishes up to

their juvenile stage, at which point the shape of their lips should be diagnostic.

Neither robust nor notchlip redhorse has reached the juvenile stage by 20 mm TL,

which means there are larval stages that cannot be identified with this key.

Although the size of the fishes when the lips become diagnostic is unknown, I

hypothesize that it is somewhere in the 30–50 mm TL range. Thus, there is a gap of

approximately 10–30 mm TL in which the fishes cannot be identified without using

the genetic techniques devised by Wirgin et al. (2004). Based on the growth rate of

the notchlip redhorse in the lab, this gap probably represents approximately 30–90

days of growth and development time.

Even if the specific traits in this key are non-diagnostic in fishes over 20 mm TL,

the general theme, that robust redhorse are more developed than notchlip redhorse

at similar size, should remain valid. Robust redhorse consistently begin to develop

each fin at a smaller size than notchlip redhorse. By 20 mm TL, robust redhorse

34

have rudimentary anal fins, sometimes with rays, and notchlip redhorse’s anal fins

are minimally developed if they are developed at all. The anal fin should continue

developing more quickly in robust redhorse, gaining a full complement of rays and a

well-defined profile at a smaller TL than in notchlip redhorse. The other characters

in the key should converge either by 20 mm TL or soon after. Any key made to

diagnose fishes beyond 20 mm TL should consider anal fin development.

There needs to be further study into how well the key works on fishes from

outside of the drainage. The key has not been tested on fishes collected outside of

the Oconee River in middle Georgia, and there could be significant local variations

in the fishes outside of Georgia that render the key inaccurate for those drainages.

When this project was conceived, robust redhorse had not been rediscovered outside

of Georgia, and including fishes from other systems would have been out of the

scope of this research. The Robust Redhorse Conservation Committee has been very

successful in finding additional populations of robust redhorse in Georgia and the

Carolinas, and the key should be tested for accuracy in these other drainages.

There are also several other closely related catostomids throughout the robust

redhorse’s range, notably the striped jumprock (Scartomyzon rupiscartes) and the

undescribed Scartomyzon species informally known as the brassy jumprock. I was

unable to obtain larval jumprocks for this key. Including them in future keys would

be interesting and worthwhile.

G.B. Fairchild once said that keys are “made by people who don’t need them for

people who can’t use them” (Wilkerson and Strickman 1990). Although that’s not

entirely true, larval fish keys are not perfect. The sources of error are too common,

and identification can be as much an art as it is a science. I have attempted to make

this key as accurate as possible by controlling the error of the entire identification

process. Hopefully, with conscientious users and good samples, this key will be a

useful tool for future research.

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Appendix A

Key for Identifying Larval Moxostoma in the Oconee River, Georgia

Note

The following is an identification key for the larvae of the Moxostoma species

suckers found in the Oconee River, Georgia: the robust redhorse, M. robustum, and

the notchlip redhorse, M. collapsum. The key does not identify larvae of either the

striped jumprock, Scartomyzon rupiscartes, or the undescribed Scartomyzon known

informally as the brassy jumprock, both of which may have a similar appearance to

the Moxostoma. The key is not separated by total length (TL), although total

length is used to help separate the fishes. The key covers fishes between 10 mm and

20 mm in total length.

Identification Key

1 Dorsal Fin Development

a. Dorsal fin not present or anterior and posterior margins not well-defined . . . . 2

b. Dorsal fin forming with anterior and posterior margins visible and

well-defined . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2(1) Total Length

a. Total length is less than 13.5 mm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

b. Total length is greater than or equal to 13.5mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

43

44

3(1) Total Length

a. Total length is less than 15.0 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. robustum

b. Total length is greater than or equal to 15.0mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4(2) Head Position

a. Head is lifted away from yolk sac . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

b. Head is curved around yolk sac . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. collapsum

5(2) Total Length

a. Total length is less than 14.0 mm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

b. Total length is greater than or equal to 14.0 mm . . . . . . . . . . . . . . . M. collapsum

6(3) Anal Fin Development

a. Anal fin development has begun, with rudimentary rays forming in some

specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. robustum

b. No obvious anal fin development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. collapsum

7(4) Total Length

a. Total length is less than 12.0 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. robustum

b. Total length is greater than or equal to 12.0 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

8(5) Digestive tract Development

a. Digestive tract developed and functional . . . . . . . . . . . . . . . . . . . . . . . . M. robustum

b. Digestive tract not functional . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. collapsum

9(7) Eye Pigmentation

45

a. Middle of eye with dark brown or black pigment . . . . . . . . . . . . . . . M. collapsum

b. Middle of eye yellowish or lacking pigment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

10(9) Total length

a. Total length less than 12.6 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. robustum

b. Total length greater than or equal to 12.6 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

11(10) Digestive tract Development

a. Digestive tract developed and functional . . . . . . . . . . . . . . . . . . . . . . . . M. robustum

b. Digestive tract not functional . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. collapsum

Appendix B

Characters Measured for the Classification Tree

The following tables contain information on the ontogenetic characters measured to

form the key. The characters measured varied with each size class (10.0–10.9 mm,

11.0–11.9 mm, and so forth); once a character appeared in a single specimen of

either species in a size class, it was measured for all specimens in that and each

successive size class until it was present in all specimens of both species, at which

point it was scored as the more developmentally advanced state for all remaining

size classes.

Table B.1: Descriptive and ontogenetic traits measured on robust redhorse (Moxos-

toma robustum) and notchlip redhorse (M. collapsum). The size-class(es) for which

the trait was measured is listed. A more detailed description of the traits appears in

Table B.2


Yolk shape 10 0.730

Yolk shape 11 < 0.001

Head position 10 0.001

Head position 11 < 0.001

Myomere development 10 0.001

Myomere development 11 0.003

Pectoral flaps 10 0.001

Continued on next page

46

47

Table B.1 — continued from previous page


Notochord flexion 10 0.001

Notochord flexion 11 < 0.001

Eye pigment 10 0.001

Body pigment 10 0.251

Pectoral fins 11 0.001

Pectoral fins 12 < 0.001

Caudal fin 11 < 0.001

Ventral finfold 11 < 0.001

Ventral finfold 12 < 0.001

Middle of eye pigment 11 0.001

Middle of eye pigment 12 < 0.001

Head pigment 11 < 0.001

Myosepta pigment 11 < 0.001

Peduncle pigment 11 0.004

Yolk depth 12 < 0.001

Branchiostegals 12 0.001

Opercular flaps 12 0.004

Caudal fin rays 12 0.004

Caudal fin differentiation 12 0.004

Yolk sac pigment 12 0.111

Peduncle pigment II 12 0.162

Peduncle pigment II 13 0.001

Yolk depth II 13 < 0.001

Nares 13 < 0.001


48



Digestive tract 13 < 0.001

Caudal fin II 13 < 0.001

Urostyle 13 0.008

Dorsal fin 13 < 0.001

Ventral finfold II 13 < 0.001

Dorsum melanophores 13 < 0.001

Dorsum melanophores 14 0.934



Chin pigment 13 0.689

Chin pigment 14 0.197

Yolk sac 14 < 0.001

Mouth position 14 0.009

Pelvic fins 14 < 0.001

Dorsal fin margins 14 < 0.001

Dorsal finfold 14 < 0.001

Anal fin 14 < 0.001

Arrow-shaped pigment 14 0.004

Arrow-shaped pigment 15 0.273

Gut melanophores 14 0.007

Jaw pigment 14 0.668

Gill arch pigment 14 < 0.001

Mouth position II 15 0.020

Anal fin II 15 0.001


49



Pelvic flaps 15 0.001

Dorsal finfold II 15 0.001

Ventral finfold III 15 0.001

Operculum length 16 835

Operculum length 20 0.157

Pelvic fins 16 0.003

Lip pigment 16 0.003

Snout pigment 17–19 0.003

Jaw pigment II 16 0.003

Head profile 16 0.003

Dorsal fin profile 20 0.157

Anal fin III 20 0.003

Pelvic fins II 20 0.157

Dorsal finfold III 20 0.157

Squamation 20 0.0157

Scale pigment 20 0.0157

Dorsum melanophores II 20 0.0157

50

Table B.2: Description of traits measured on larval robust redhorse (Moxostoma

robustum) and notchlip redhorse (M. collapsum). Traits were measured on fishes of

all sizes. Traits were scored as the more advanced state for all remaining size classes

once the trait became present in all specimens in a given size class. In cases where

the trait has a more and less advanced state, the less advanced state is listed first.

Character Description

Yolk shape Bulbous: yolk sac bulbous anteriorly

Cylindrical: yolk sac cylindrical throughout

Head position Curved: head still curved

Lifted: head lifted away from yolk sac

Myomeres Ongoing: post-anal myomeres still developing

Complete: all post-anal myomeres fully developed

Pectoral flaps Absent: pectoral flaps absent

Present: pectoral flaps present

Notochord flexion Straight: tip of notochord not flexed

Flexed: tip of notochord flexed

Eye pigment Unpigmented: middle of eye yellowish or unpigmented

Pigmented: middle of eye with brown or black pigment

Body pigment Absent: body without any pigmentation

Present: some melanophores present on body

Pectoral fins Absent: pectoral flaps present or pectoral fins absent

Present: pectoral fins (not flaps) present

Caudal fin Undifferentiated: caudal fin undifferentiated from body

Differentiated: caudal fin differentiation has begun

Ventral finfold Absent: ventral finfold absent


51



Present: finfold present on posterior margin of yolk sac

Eye pigment II Absent: middle of eye unpigmented

Present: middle of eye with brown or black pigment

Head pigment Absent: Head pigment absent

Present: Pigment scattered on head over brain

Myosepta pigment Absent: pigment absent on median myosepta

Present: dashed line of pigment along median myosepta

Peduncle pigment Absent: caudal peduncle unpigmented

Present: Pigment scattered at base of caudal peduncle

Yolk depth Deep: yolk 2x deeper than torso

Shallow: yolk depth approximately equal to torso depth

Branchiostegals Absent: branchiostegals absent

Present: branchiostegal development is visible

Opercular flaps Absent: opercular flaps absent

Present: opercular flaps visible or forming

Caudal fin II Absent: caudal fin rays absent

Present: caudal fin rays present or forming

Caudal fin III Absent: caudal fin undifferentiated from body

Present: caudal fin obviously differentiated from body

Ventral finfold II Absent: ventral finfold absent anterior of yolk

Present: finfold present from yolk to pectoral fins

Yolk sac pigment Absent: yolk sac unpigmented

Present: pigment on dorsal margin of yolk sac

Peduncle pigment II Absent: caudal peduncle pigment sparse or light


52



Present: Dark pigment outlines caudal peduncle

Yolk depth II Deep: yolk depth greater than torso depth

Shallow: yolk depth less than torso depth

Nares Absent: nares absent

Present: nares visible

Digestive tract Functional: digestive tract fully developed

Not functional: digestive tract development incomplete

Caudal fin IV Unilobed: caudal fin unilobed

Bilobed: caudal fin bilobed

Urostyle Long: urostyle extends to dorsal margin of caudal fin

Short: urostyle stops before dorsal margin of caudal fin

Dorsal fin Absent: dorsal fin and fin profile absent

Developing: developing dorsal fin or fin profile evident

Ventral finfold III Absent: ventral finfold absent

Present: ventral finfold present

Dorsum pigment Present: 3–4 melanophores between eyes and pectorals

Absent: pigment absent between eyes and pectoral fins

Chin pigment Present: 2–3 melanophores present on chin

Absent: chin melanophores absent

Yolk sac Present: some yolk sac remaining

Absent: yolk sac fully absorbed

Mouth position Terminal: mouth terminal

Subterminal: mouth position ventral and subterminal

Pelvic fins Absent: pelvic fins and flaps absent


53



Present: pelvic fins or flaps present

Dorsal fin margins Undefined: margins of dorsal fin undefined

Defined: anterior and posterior margins well-defined

Dorsal finfold Continuous: finfold present throughout dorsum

Absorbed: finfold restricted to posterior half of dorsum

Anal fin Absent: anal fin absent; development has not begun

Present: anal fin development has begun

Arrow-shaped pigment Absent: pattern absent

Present: pattern present anterior to pectoral fin base

Gut melanophores Absent: melanophores absent on gut

Present: melanophores on gut posterior to air bladder

Jaw pigment Absent: pigment absent on jaw

Present: margin of jaw outlined in pigment

Gill arch pigment Absent: pigment absent on gill arches

Present: gill arches outlined in pigment

Mouth position II Oblique: mouth subterminal and oblique

Horizontal: mouth subterminal and horizontal

Anal fin II Early: anal fin absent or in nascent stages

Late: anal fin developing, possibly with rays

Pelvic flaps Absent: pelvic fin development has not begun

Flaps: pelvic flaps developed

Dorsal finfold II Present: significant portion of finfold still remains

Reduced: finfold greatly reduced or absent

Ventral finfold IV Present: significant portion of finfold still remains


54



Reduced: finfold greatly reduced or absent

Operculum length Short: operculum short of fin base

Long: operculum extends to fin base

Pelvic fins Short: fins short of margin of ventral finfold

Long: fins extend to margin of ventral finfold

Lip pigment Absent: pigment absent on upper lip

Upper: pigment present on upper lip

Snout pigment Absent: melanophores absent across snout

Bar: bar of small melanophores present across snout

Jaw pigment II Light: minimal or no pigment on jaw

Heavy: jaw outlined in heavy pigment

Head profile Flat: head profile flat posterior to eyes

Concave: head profile slightly concave posterior to eyes

Dorsal fin profile Straight: dorsal fin profile straight

Concave: dorsal fin profile concave

Anal fin III Early: fin without distinct profile and rays

Late: fin with distinct profile and 7–8 rays

Pelvic fins II Early: pelvic fins less developed, fewer than 7 rays

Developed: fins well-developed with at least 7 rays

Dorsal finfold III Present: finfold still remains

Absent: finfold entirely absorbed

Squamation Absent: squamation absent on caudal peduncle

Present: squamation visible on caudal peduncle

Scale pigment Absent: scales unpigmented


55



Present: pigment outlines scales from head to caudal fin

Dorsum pigment II Band: 3–4 melanophores from occiput to dorsal fin

None: band of melanophores absent

Appendix C

Development of Young Robust Redhorse

The following is a description of the development of larval and early juvenile robust

redhorse (Moxostoma robustum) based on a report prepared by Robert Wallus of

Murphy, North Carolina. This narrative is based on laboratory-reared specimens

from the Oconee River, Georgia and was used as the basis for the quantitative

analysis of the descriptive characters used in the model fitting (described in Chapter

3, Methods). Terminology is used as defined in Wallus et al. (1990).

Morphology

7.2–8.1 mm TL (newly hatched): Fish in this range have a yolk sac that is large and

bulbous (almost round) anteriorly. The head is small and slightly curved around the

anterior end of the bulbous portion of the yolk sac. Myomere development is

incomplete.

9.7–10.5 mm TL: The yolk material is visibly reduced and is cylindrical or

tubular throughout its length. The head has lifted away from the yolk. The

stomodeum is forming, but the mouth is not open.

11.2–11.7 mm TL: The yolk sac is still tubular, but it tapers in thickness

posteriorly. Yolk depth is greater than the depth of the torso. Internal head

development is visible; the mouth opening is apparent and gill arches are forming.

The heart is developing just anterior to the yolk sac.

56

57

12.2–12.9 mm TL: The yolk is reduced but still tubular, and is approximately as

deep as the torso. Branchiostegal formation has begun and opercular flaps are

forming.

13.0–14.0 mm TL: Opercular development continues until the opercular flap

covers the gills. Nares are visible and the otic chamber has formed. The head

appears flattened in profile and the eyes appear slightly flattened. The mouth is

subterminal and oblique. The digestive tract is functional on some individuals by

13.6 mm TL. The remaining yolk is still tubular at 13 mm TL and the depth of the

yolk is equal to about half the depth of the torso. The yolk is completely absorbed

son some individuals of 14.0 mm TL, but still present on others as large as 14.3 mm

TL.

14.3–16.0 mm TL: The head is flattened ventrally. The mouth is ventrally

located and progresses from subterminal and oblique to subterminal and horizontal.

The operculum is present to the base of the pectoral fins by 16 mm TL.

18.6–20.0 mm TL: Development continues and the dorsal head profile is now

slightly concave posterior to the eyes.

20.3 mm TL: Morphological development continues. Squamation is visible on

the caudal peduncle.

21.9–22.5 mm TL: Scalation is now visible mid-laterally on the body from the

caudal peduncle to the head.

Fin Development

72.–8.1 mm TL: The median finfold begins dorsally near the middle of the body

and extends posteriorly around the notochord, ending ventrally at the posterior

margin of the yolk sac. No other fin development is apparent.

58

9.7 mm TL: Pectoral flaps are present. The dorsal origin of the median finfold is

set back about 25% of TL from anterior edge of snout. The tip of the notochord is

slightly flexed.

11.2–11.7 mm TL: Notochord flexion is still slight. Caudal fin differentiation is

beginning: rays are unformed, but basal elements of the hypural complex are

forming. The developing pectoral fins are about 0.5 mm long. The dorsal origin of

the median finfold is now around myomeres 8–10; ventrally, the finfold is beginning

to form on the posterior margin of the yolk sac.

12.2–12.9 mm TL: Notochord flexion is more obvious and basal elements of the

caudal fin are well-formed. Incipient rays are forming in the caudal fin: 8–12 rays are

visible by 12.9 mm TL. The ventral finfold is now present anteriorly on the yolk sac

to about the position of the pectoral fins. The dorsal profile of the median finfold is

beginning to elevate at the future position of the dorsal fin.

13.6–14.3 mm TL: Pectoral fins are about 1.25 mm long. The caudal fin is

becoming bilobed with the urostyle extending to the dorsal margin of the caudal fin.

The dorsal fin profile is forming in the dorsal finfold, which is much reduced

anteriorly. Differentiation in the forming dorsal fin is obvious on some individuals by

13.8 mm TL. The anterior and posterior margins of the dorsal fin are nearly defined

between 14.0 and 14.3 mm TL and incipient rays are forming. The ventral finfold is

also decreasing in width and extends anteriorly to a position near the middle of the

abdominal cavity. Pelvic fins appear between 14.0 and 14.3 mm TL as narrow flaps

positioned ventro-laterally beneath the anterior half of the developing dorsal fin and

at the juncture of the gut and torso.

14.3–14.5 mm TL: The caudal fin is distinctly bilobed and well-developed, with

18 primary rays, some of which are segmented. The urostyle, positioned

immediately dorsal to the anterior-most primary caudal ray, still extends beyond

the hypural plate. The dorsal fin origin is around myomere 12 or 13, and the dorsal

59

fin has visible rays and well-defined anterior and posterior margins. The remainder

of the dorsal finfold is restricted between the dorsal fin and caudal fin, and is less

than half as deep as the torso. The ventral finfold is also reduced: it extends

anteriorly to the anus to near the pectoral fin bases at about the point of greatest

body depth. The anal fin is forming with pterygiophores (but no rays) present by

14.3 mm TL. The pectoral fins are about 1.5 mm long with visible rays.

15.2–15.9 mm TL: Anal fin rays are forming. There are 10–11 rays present in

the developing dorsal fin. The pelvic flaps are about half the width of the remaining

ventral finfold. The urostyle still extends past the hypural plate.

16.0–16.9 mm TL: The anal fin has a rounded margin, defined insertion, and

5–6 visible rays. The pelvic fins, with visible rays, extend to the margin of the

remaining ventral finfold. A small amount of dorsal finfold is still present between

the dorsal and caudal fins. The ventral finfold is present from the anus anteriorly to

about midway between the pectoral and pelvic find.

17.7–19.2 mm TL: No dorsal finfold remains. The ventral finfold is restricted to

the area between the pelvic fins and the anus. Fin development appears to be

nearing completion: all fins have well-developed rays and defined margins. The

urostyle still extends beyond the margin of the hypural plate. the dorsal fin profile

appears concave with at least 13 visible rays. The anal fin has 7–8 rays and eight or

more rays are visible in the pelvic fins. The pectoral fins are well-developed with at

leas 12–14 rays present.

20.0–22.5 mm TL: A very small remnant of finfold is present immediately

anterior to the anus at 20.3 mm TL. The finfold is completely gone and fin

development is complete, or nearly so, by about 22.5 mm TL.

60

Pigmentation

7.2–8.1 mm TL: The eyes, head, and body are all lacking pigment. The yolk is

yellowish in color.

9.7–10.5 mm TL: Eye pigment is becoming apparent. The only body pigment

consists of thin, dark dashes on some specimens along the median myosepta dorsal

to the yolk sac.

11.2–11.7 mm TL: The eyes are dark brown. Dorsally, pigment is scattered on

the head over the brain, narrowing on the occiput to a single mid-dorsal row on the

body to the origin of the dorsal finfold. Scattered melanophores are present dorsally

and ventrally at the base of the finfold on the caudal peduncle. Melanophores are

present mid-ventrally on the yolk sac from the base of the pectoral fins to the anus.

Lateral pigment consists of a dashed line along the median myosepta from the head

to about the middle of the caudal peduncle.

12.2–12.9 mm TL: The eyes are dark brown or black. The dorsal pigment

described above now consists of large, dark melanophores. Indistinct rows of small

melanophores appear on either side of the dorsal finfold in the middle of the body.

The pigment outlining the caudal peduncle is now darker. Internal pigment appears

scattered on the dorsal margin of the yolk sac.

13.0–14.3 mm TL: In addition to previously described pigment patterns,

melanophores are now present on the head around the tip of the snout at the

anterior margins of the nares. A few large melanophores are now scattered dorsally

on the head between the eyes. There are distinct lateral rows of pigment dorsally

over the middle of the body. These rows of pigments fuse posteriorly with dense,

scattered pigment on the caudal peduncle. A row of 3–4 melanophores is present

ventral to the otic chamber on the side of the head between the eyes and pectoral

fins. This row of pigment curves downward anteriorly from about the height of the

dorsal margin of the pectoral fin base. Ventral pigment on the yolk sac is now a

61

wide band of melanophores. Internally, the dorsal margin of the abdominal cavity is

covered with melanophores. Pigment is scattered on the caudal fin and at the base

of the caudal fin by 14.0–14.3 mm TL. Two to three melanophores are present on

the chin in some individuals.

14.3–15.9 mm TL: Ventral pigment is still heavy. There is an arrow-shaped

pigment pattern anterior to the pectoral base with its point near the isthmus. This

scattered pigment narrows at the base of the pectoral fins to a double row of

melanophores, which extend posteriorly to about the anterior margin of the ventral

finfold. Heavy pigment is scattered at the base of the finfold to the anus. Chin

pigment is present. Pigment outlines the gill arches. Pigment also is present on the

upper lip and snout. Internally, melanophores are scattered dorsally on the gut

posterior to the air chambers.

16.0–16.9 mm TL: Dorsally, uniformly scattered pigment covers the head,

occiput, and otic chamber. The large melanophores over the brain and in the single

row from the occiput to the dorsal fin origin are still present. Scattered small

melanophores cover the remainder of the dorsum. From the dorsal fin origin to the

middle of the caudal peduncle this pigment consists of scattered pigment between

dorso-lateral rows of small melanophores. From the middle of the caudal peduncle

to the base of the caudal fin this pigment consists of dark, densely scattered

pigment. Lateral pigmentation is little changed. Small melanophores are scattered

around the snout. The upper lip is pigmented on all specimens and small

melanophores are present on the lower lip in some specimens. Pigment is still visible

on the chin. There are fewer melanophores on the ventrum, especially on the gut

anterior to the developing pelvic fins. Dark, tightly scattered pigment is present on

the ventral caudal peduncle between the anal fin and the caudal fin. The arrow

pattern anterior to the pectoral fin bases is still present.

62

17.0–19.2 mm TL: Pigmentation in the form of small melanophores is beginning

to expand dorso-laterally. By 18.3 mm TL, little pigment is present ventrally

anterior to the pelvic fins, but there is still dark, tightly-scattered pigment posterior

to the pelvic fins along the gut and posterior to the anal fin. Small melanophores

are scattered throughout the caudal fin and on the anterior half of the dorsal fin.

Some pigment is present on the anal fin by 19.0 mm TL.

19.2–22.5 mm TL: The lateral progression of pigment is continuing. At 19.2 mm

TL, pigment is scattered laterally to just above the median myosepta. By 20.3 mm

TL, pigment has progressed slightly pas the median myosepta on sides of the body

anterior to the anal fin and small melanophores outline the scales on the caudal

peduncle. The scales are outlined in pigment from the caudal fin to the head on

individuals 21.9–22.5 mm TL.

Appendix D

Development of Young Notchlip Redhorse

The following is a description of the development of larval and early juvenile

notchlip redhorse (Moxostoma collapsum) based on a report prepared by Robert

Wallus of Murphy, North Carolina. This narrative is based on laboratory-reared

specimens from the Broad River, Georgia and was used as the basis for the

quantitative analysis of the descriptive characters used in the model fitting

(described in Chapter 3, Methods). Terminology is used as defined in Wallus et al.

(1990).

Morphology

9.0–9.3 mm TL (recently hatched): Yolk is bulbous anteriorly and cylindrical or

tubular posteriorly. Yolk is large: its greatest depth at least twice that of the torso.

Head is small and curved around the anterior margin of the yolk sac. Internal

development of the head is beginning: the developing brain and otic capsule are

visible. Post-anal myomeres are still developing.

10.5 mm TL: The head is lifting off the yolk, which is now bulbous anteriorly

and tubular behind.

11.2–11.9 mm TL: Yolk remains slightly bulbous anteriorly, otherwise tubular.

At 11.2 mm TL, the head is curved around the anterior margin of the yolk sac, but

by 11.9 mm TL the head has lifted and is on an axis nearly parallel to that of the

body. Head development continues with developing brain and otic capsule visible;

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stomodeum development begins and the mouth is open on some specimens by 11.9

mm TL. Gill development begins. Post-anal myomere development continues.

12.1–12.7 mm TL: On most specimens the yolk is tubular throughout its length;

yolk depth is about twice that of torso. The head is on the same axis as the body.

Mouth opening may not have developed on all individuals but appears open at 12.7

mm TL. Developing gills are visible. By 12.7 mm TL, developing heart is apparent

just anterior to yolk and nasal openings are visible anterior to the eyes.

13.0–13.9 mm TL: Yolk remains tubular along its length, but absorption varies

with individual depth ranging from about equal to about twice that of the torso

(yolk depth was still about twice torso depth on an individual of 13.9 mm TL). Eyes

appear flattened. Gill development continues: opercular flap appears by 13.4 mm TL

and covers about half of the gill chamber by 13.8 mm TL. The mouth appears

subterminal and oblique by 13.9 mm TL.

14.0–14.9 mm TL: Yolk sac generally tubular along its length from gular region

to anus. Amount of yolk remaining ranges from about twice the depth of the torso

in an 14.2 mm TL individual to 14.1 mm TL individuals with very little yolk

remaining. Depth of yolk approximately to torso depth for a 14.7 mm TL

individual, so yolk may persist on larger specimens. However, all specimens greater

than 14.7 mm TL had completed yolk absorption. In summary, the yolk-sac phase of

these specimens is complete between about 14.2 and 14.8 mm TL. The last remnant

of remaining yolk is present just posterior to the air bladder. The gut appears above

the diminishing yolk; by 14.8 mm TL, the digestive tract is functional. The eyes still

appear slightly flattened. Development of the opercle, mouth, nasal openings, gills,

and heart is apparent. The opercle nearly covers the gill chamber on some

individuals by 14.0 mm TL and completely covers it on a 14.7 mm TL individual.

Branchiostegal development begins and is well-defined by 14.7 mm. Developing air

bladders are visible by 14.3 mm TL, their positions marked by concentrations of

65

pigment above them. Gill arches are evident by 14.3 mm TL and gills are

well-developed and visible by 14.7 mm TL. At 14.3 mm TL, the mouth is nearly

terminal and oblique with the lower lip at the level of the ventral margin of the eye;

however, a 14.9 mm TL individual’s mouth appeared subterminal and oblique.

15.1–15.6 mm TL: Internal development continues. Eyes appear flattened.

Mouth is subterminal and slightly oblique.

16.0–17.3 mm TL: Eyes appear slightly flattened. Mouth is subterminal, ventral,

and parallel to the body axis.

20.8–21.0 mm TL: Mouth is ventral and subterminal. Eyes appear round.

Fin Development

9.0–9.3 mm TL (recently hatched): The median finfold originates dorsally at a point

about 38% of TL and is present posteriorly around the notochord and ventrally to

the posterior margin of the yolk sac. No other fin development is apparent.

10.5 mm TL: The anterior dorsal finfold origin is at approximately 31% of TL.

11.2–11.9 mm TL: The finfold is little changed: it’s origin is at about 30% of TL

(around myomere 10–11). Pectoral fins visible as flaps by 11.3 mm TL.

12.1–12.7 mm TL: The dorsal origin of the finfold is at 30–34% of TL (myomere

10–11). The finfold becomes more visible on the posterior margin of the yolk sac.

The pectoral flaps are about 0.3 mm long at 12.1 mm TL.

13.0–13.9 mm TL: The dorsal finfold origin is at myomere 9–11, or about

1/4–1/3 of TL. As the yolk diminishes, the finfold becomes visible ventrally on the

yolk sac, extending anteriorly to the base of the pectoral fins by 13.7 mm TL.

Flexion begins; the urostyle moves dorsally to about a 45◦ angle by 13.9 mm TL.

Elements of the developing hypural plate are visible by 13.7 mm TL and caudal fin

ray development begins posterior to the upturned urostyle. 11–12 rudimentary rays

are visible in the caudal fin by 13.8 mm TL. An undulation in the dorsal finfold is

66

apparent in the area of the future dorsal fin. Pectoral fins are about 0.5 mm long at

13.9 mm TL.

14.0–14.9 mm TL: Fin development varies on individuals throughout this length

range. The dorsal finfold origin is usually at myomere 10 or 11. The occurrence of

the finfold ventrally on the yolk sac is variable and seems related to the amount of

yolk absorption. On a late yolk-sac larva (14.3 mm TL), the finfold was prominent

dorsally and ventrally, with its greatest depth dorsally above the anus and its

greatest depth ventrally between the anus and the posterior margin of the air

bladder. Dorsal fin develops with six pterygiophores and 3–4 rays visible by 14.9

mm TL. Pectoral fin growth and development also seems unrelated to TL. Rays

become visible as early as 14.3 mm TL. In this length range the length of the

pectoral fins varies from about 0.8 mm to about 1.75 mm. Tissue differentiation

becomes visible in the area of the future anal fin by 14.7 mm TL.

Caudal fin development also varies, but it progresses throughout the size range.

Flexion is obvious on most individuals, but hypural development and formation of

caudal fin rays varies without relation to TL. An 14.5 mm TL individual had no

visible caudal fin rays, while a late yolk-sac larva (14.1 mm TL) had 14–16

segmented rays. Flexion exceeded 45% on a 14.4 mm individual, but there was

minimal caudal fin development. By 14.9 mm TL, 16–18 segmented caudal fin rays

are visible, and hypural elements are well-defined. During caudal fin development,

the shape of the fin varies, progressing from a pointed finfold to a rounded tail that

widens posteriorly late in the yolk-sac phase and finally appears squared-off

posteriorly with visible lobes (at 14.9 mm TL). At 14.9 mm TL, the tip of the

urostyle extends beyond the dorsal profile of the torso.

15.1–15.6 mm TL: Finfold is little changed. The caudal fin appears squared-off

with obvious dorsal and ventral lobes; 15–17 segmented rays are present, and

hypural development is well defined. The tip of the urostyle still extends past the

67

dorsal profile of the torso. At 15.6 mm TL, the dorsal fin has eight pterygiophores

and seven visible rays, and is elevated to a point at the tip of its longest ray.

16.0–16.5 mm TL: Notches form in the finfold at several places: the posterior

margin of the developing dorsal fin, between the dorsal finfold and the caudal fin,

and at the posterior margin of the future anal fin. Caudal fin development varies: a

16.0 mm TL individual had advanced caudal development (17–18 segmented rays

and a bilobed appearance), but a 16.5 mm TL specimen ad only 13 segmented rays

and a rounded fin profile with no apparent lobes. The dorsal fin profile is pointed at

the tip of its longest ray with as many as 10–11 pterygiophores and rays visible.

Anal fin margins become defined and rudimentary rays form. The pelvic fins appear

as ventro-lateral flaps on the body below the middle of the dorsal fin.

17.1–17.3 mm TL: The finfold now has clearly defined notches at the posterior

margin of the dorsal fin, between the dorsal finfold and the caudal fin, and at the

posterior margin of the anal fin. At 17.3 mm TL, the caudal fin is bilobed and

almost completely developed, with 18 segmented rays and 3–4 secondary rays. The

tip of the urostyle is no longer visible above the dorsal profile of the torso. The

dorsal fin had 13 pterygiophores and 11–12 rays. Three pterygiophores and 3–4 rays

are visible in the developing anal fin. The pelvic flaps, positioned at the posterior

margin of the air bladder, are about 1/2 the depth of the ventral finfold.

20.8 mm TL: The finfold is greatly reduced dorsally and ventrally: a dorsal

remnant is barely visible between the dorsal and caudal fins and the ventral

remnant is present from anterior to the pelvic fins posterior to the anus; ventrally,

the finfold is completely absent between the anus and the caudal fin. The caudal fin

is bilobed with 18 segmented rays and 7–8 secondary rays. The posterior margin of

the anal fin is defined with 8 pterygiophores and 7 rays visible. The pectoral fins are

well-developed with at least 12 rays. The dorsal fin has 14 rays. Pelvic fins are about

1.25 mm long and extend past the margin of the ventral finfold with 6–7 visible rays.

68

17.7 mm SL (21.0+ mm TL: The only remaining remnant of the finfold is

present ventrally between the pelvic fins and the anus. Fin development for all fins

in nearly complete. Fin rays are as follows: the pectoral fins have 11–12 rays, the

anal fin has 7, the dorsal fin has 14, and the pelvic fins have 6–7 rays. The distal half

of this specimen’s caudal fin was missing, but its development appears complete.

Pigmentation

9.0–10.5 mm TL: The head and body lack pigment. The middle of the eye and the

yolk are yellowish.

11.2–11.9 mm TL: At 11.2 mm TL, the body and the head are unpigmented and

the middle of the eye and yolk are still yellowish. By 11.9 mm TL, the eyes are

developing pigment. Dorsal pigment is limited to a few melanophores in a row

anterior to the finfold origin. There is an irregular row of melanophores laterally

along the median myosepta from the head to the posterior margin of the yolk sac.

There is pigment scattered mind-ventrally on the yolk sac.

12.1–12.7 mm TL: The eyes become dark. At 12.2 mm TL, a few melanophores

are present over the hindbrain and occiput. Two large melanophores are present

mid-dorsally between the occiput and the dorsal finfold origin. Pigment is scattered

mid-ventrally on the yolk and a row of melanophores is present along the median

myosepta from the head to mid-body.

By 12.7 mm TL, there are many scattered melanophores on the dorsum from the

head above the eyes to the occiput. Along the body, pigment is scattered

mid-dorsally, near the base of the finfold, almost to the end of the urostyle. Ventral

pigment is concentrated on the yolk and along the base of the finfold onto the

caudal peduncle. Mid-lateral pigment is present along the median myosepta form

the pectoral fin flaps posteriorly to the mid caudal peduncle. Internal pigment if

69

visible in the gular region over the developing heart and along the ventral margin of

the abdominal cavity over the yolk.

13.0–13.9 mm TL: The eye is fully pigmented. Pigmentation patterns are

basically as described above with a few advancements. Dorsally, large melanophores

cover the head from the middle of the eyes to over the occiput. Pigment becomes

densely concentrated dorsally and ventrally along the caudal peduncle. Some

melanophores are visible around the tip of the urostyle. The mid-lateral row of

pigment now consists of 1–2 melanophores on each myomere. Ventral pigment on

the yolk appears as two rows of large melanophores positioned mid-ventrally.

Pigment appears on the tip of the snout. Internal pigment over the yolk expands

anteriorly, forming a broad patch of pigment over the area of the future air bladder.

Some pigment cells are scattered dorso-laterally along the yolk sac. Scattered

melanophores appear laterally on the developing hypural complex and in the region

of the developing caudal fin rays (the latter is only visible with a polarized light

filter).

14.0–14.9 mm TL: The pigment patterns described above progress with the

following observations. The tip of the snout becomes covered with a concentration

of small melanophores (which appear as a bar of pigment across the snout) and the

margin of the upper jaw becomes outlined in pigment. Dorsally, large melanophores

are present on the head between the eyes, over the brain, between the otic

chambers, and over the occiput. On the body, a mid-dorsal row of pigment is

flanked by irregular rows of melanophores, which gives the appearance of three rows

along the back. Pigment is lacking mid-dorsally only in the region of the developing

dorsal fin. Posterior to the anus, melanophores are smaller, concentrated across the

dorsum, and extended posteriorly almost to the urostyle. Similar dense pigment is

present ventrally behind the anus. Viewed laterally, these heavy concentrations of

pigment, dorsally and ventrally, outline the caudal peduncle.

70

There is a row of 2–4 melanophores parallel to the body axis on the chin. The

developing branchiostegals have a few small melanophores scattered on them. The

ventral gular pigment consists of scattered melanophores that are present from the

base of the pectoral fins to a point at the isthmus. A mid-ventral row of 5–6 pairs of

large melanophores extends posteriorly from the base of the pectoral fins and joins a

single row of large melanophores that extends to the anus. This row of pigment,

posterior to the pairs of melanophores, is usually a single row. However, more than

one melanophore may be present mid-ventrally on some individuals.

Laterally, the most obvious body pigment is restricted to the single row of small

melanophores (2–4 per myomere) that extends from the pectoral fins onto the

caudal peduncle. Some scattered pigment is present on the developing hypural

complex, caudal fin, and on the urostyle near the tip. A few small melanophores are

present on the side of the head, lateral to the otic chamber and on the opercle.

Internally, 3–4 melanophores appear in a diagonal row at the base of the

pectoral fins. The gill arches become outlined with pigment. Large melanophores are

scattered over the developing stomach, behind the eyes, and under the brain. Dense

pigmentation covers the developing air bladders and extends posteriorly along the

ventral surface of the abdominal cavity and along the dorsal surface of the gut.

15.1–15.6 mm TL: Pigment is as above with a few advancements. Laterally,

about midway between dorsal pigment and the mid-lateral stripe is an internal row

of small melanophores that extends from the posterior margin of the hindbrain to

the upturned urostyle. By 15.6 mm TL, a few scattered melanophores are present

ventro-laterally along the posterior 1/3 of the gut.

16.0–16.5 mm TL: Pigment is as above with a few advancements. On the body,

pigment becomes scattered across the entire dorsal surface except mid-dorsally

under the developing dorsal fin. Some melanophores are slightly dorso-lateral in

position. Dorsal head pigment between the eyes extends anteriorly to the bar of

71

pigment across the snout. Scattered pigment increases on the head laterally and

ventrally.

17.1–17.3 mm TL: Pigment is generally the same as above. External lateral

pigment is present on the stomach and air bladders.

20.8 mm TL–17.7 mm SL: Pigment is as above with several additions. Dorsal

pigment starts at the mouth and is prominent over the snout onto the head. The

head is covered with pigment dorsally and dorso-laterally to about the middle of the

eyes. A band of pigment 3–4 cells across extends mid-dorsally from the occiput

pigment to the dorsal fin origin. A double row of pigment is present mid-dorsally

from the dorsal fin insertion to the caudal fin. Scattered pigment is present

dorso-laterally throughout the body. Pigment is scattered laterally along the sides of

the stomach, air bladders, and gut. Small melanophores are scattered ventrally on

the head and under the eyes. Gill arches are still outlined with pigment.

Melanophores are present along the margins of most dorsal fin rays and blotches of

pigment are present on the membranes between the rays at the distal margin of the

fin. Melanophores also outline caudal fin rays and a few are present in the anal fin.

Appendix E

Morphometric and Descriptive Measurements

The following table contains all of the measurements used to fit the classification

tree model. All measurements were made using a stereo dissecting microscope at

10x magnification. Morphometrics were measured as described in Chapter 3

(Methods). An explanation of the descriptive characters can be found in Table B.2.

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Table E.1: Morphometric and descriptive character measurements made on robust

redhorse (Moxostoma robustum), abbreviated mr, and notchlip redhorse (M. col-

lapsum), abbreviated mc. Morphometrics were measured as described in Chapter

3 (Methods) and are given in millimeters. They include total length (TL), standard

length (SL), pre-anal length (PaL), head length (HL), eye diameter (ED), pre-dorsal

fin length (PdF), and greatest body depth (GBD). Descriptive characters were scored

in one of three ways: present or more advanced (p); absent or less advanced (a); or

unmeasured (u). An explanation of the descriptive characters can be found in Table

B.2 .

Sample # Species TL SL PaL HL ED PdF GBD Yolk shape Head position Myomeres Pectoral flaps Notochord flexion Eye pigment

mc1 mc 9 8.8 7.2 1.4 0.5 3.5 na a a a a a a

mc2 mc 9.3 9.1 7.6 1.4 0.5 3.4 na a a a a a a

mc3 mc 10.5 10.3 8.5 1.3 0.5 3.3 na a a a a a a

mc4 mc 11.2 11.0 9.2 - - - na a a a p a p

mc5 mc 11.3 11.1 9.3 1.4 0.6 3.6 na a a a p a p

mc6 mc 11.8 11.5 9.4 - - - na a a a p a p

mc7 mc 11.9 11.6 9.5 1.5 0.7 - na a a p p a p

mc8 mc 11.9 11.6 9.5 1.5 0.7 3.6 na a a p p a p

mc9 mc 12.1 11.9 10.0 1.5 0.7 4.1 na p p p p p p

mc10 mc 12.2 12.0 10.1 1.6 0.7 3.6 na p p p p p p

mc11 mc 12.5 12.2 10.2 1.7 0.7 4.0 na p p p p p p

mc12 mc 12.7 12.3 10.3 1.9 0.8 4.3 na p p p p p p

mc13 mc 13 12.7 10.3 1.8 0.8 3.6 na p p p p p p

mc14 mc 13.4 13.0 10.2 1.6 0.7 3.4 na p p p p p p

mc15 mc 13.5 13.2 10.6 1.8 0.8 3.8 na p p p p p p

mc16 mc 13.5 13.2 10.7 1.9 0.8 3.7 na p p p p p p


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Table E.1 — continued from previous pageSample # Species TL SL PaL HL ED PdF GBD Yolk shape Head position Myomeres Pectoral flaps Notochord flexion Eye pigment

mc17 mc 13.5 13.1 10.7 1.8 0.8 4.3 na p p p p p p

mc18 mc 13.7 13.2 10.8 1.8 0.8 4.0 na p p p p p p

mc19 mc 13.7 13.2 10.1 2.1 0.8 4.5 na p p p p p p

mc20 mc 13.7 13.1 10.1 2.1 0.8 4.2 na p p p p p p

mc21 mc 13.8 13.4 10.7 1.8 0.9 4.3 na p p p p p p

mc22 mc 13.8 13.1 10.2 2.1 0.9 4.5 na p p p p p p

mc23 mc 13.8 13.0 10.0 2.3 0.8 4.6 na p p p p p p

mc24 mc 13.9 13.5 10.6 1.9 0.8 4.3 na p p p p p p

mc25 mc 13.9 13.3 10.2 2.0 0.9 4.5 na p p p p p p

mc26 mc 13.9 13.2 10.4 2.1 0.8 4.6 na p p p p p p

mc27 mc 14 13.5 10.8 1.8 0.8 4.3 na p p p p p p

mc28 mc 14 13.3 10.6 2.0 0.9 4.8 na p p p p p p

mc29 mc 14 13.4 10.2 2.2 0.9 4.6 na p p p p p p

mc30 mc 14.1 13.8 11.1 1.9 0.8 4.0 na p p p p p p

mc31 mc 14.1 13.3 10.1 2.6 1.0 5.6 na p p p p p p

mc32 mc 14.1 13.2 10.2 2.5 1.0 - na p p p p p p

mc33 mc 14.2 13.8 11.1 1.8 0.8 4.2 na p p p p p p

mc34 mc 14.2 13.8 11.1 1.8 0.8 4.2 na p p p p p p

mc35 mc 14.2 13.8 11.0 1.9 0.8 4.0 na p p p p p p

mc36 mc 14.2 13.4 10.4 2.1 0.8 4.6 na p p p p p p

mc37 mc 14.2 13.7 10.7 2.2 0.9 4.6 na p p p p p p

mc38 mc 14.2 13.6 10.8 2.2 0.9 4.2 na p p p p p p

mc39 mc 14.2 13.3 10.3 2.4 0.9 4.6 na p p p p p p

mc40 mc 14.2 13.5 10.5 2.1 0.9 4.9 na p p p p p p

mc41 mc 14.3 13.5 10.4 2.2 0.8 4.7 na p p p p p p

mc42 mc 14.3 13.2 10.1 2.1 0.9 4.5 na p p p p p p

mc43 mc 14.3 13.8 10.5 2.4 1.0 4.7 na p p p p p p

mc44 mc 14.3 13.7 10.6 2.5 1.0 5.0 na p p p p p p

mc45 mc 14.4 13.8 10.7 2.3 0.9 4.7 na p p p p p p

mc46 mc 14.4 13.7 10.7 2.2 0.9 4.4 na p p p p p p

mc47 mc 14.5 13.9 10.8 2.4 0.8 4.7 na p p p p p p

mc48 mc 14.5 13.5 10.4 2.3 0.9 4.8 na p p p p p p

mc49 mc 14.6 14.0 11.0 2.2 0.9 4.7 na p p p p p p

mc50 mc 14.6 14.0 10.8 2.3 0.9 4.5 na p p p p p p


75


mc51 mc 14.6 13.7 10.7 2.4 0.9 4.8 na p p p p p p

mc52 mc 14.6 13.9 10.7 2.6 1.0 5.0 na p p p p p p

mc53 mc 14.7 14.0 11.0 2.3 0.9 4.7 na p p p p p p

mc54 mc 14.7 13.7 10.5 2.4 1.0 4.9 na p p p p p p

mc55 mc 14.7 13.4 10.5 2.2 0.9 4.7 na p p p p p p

mc56 mc 14.7 13.9 10.6 2.3 0.9 4.8 na p p p p p p

mc57 mc 14.7 13.9 10.8 2.7 0.9 5.5 na p p p p p p

mc58 mc 14.8 13.5 10.6 2.5 1.0 5.0 na p p p p p p

mc59 mc 14.9 13.3 10.5 2.5 1.0 4.9 na p p p p p p

mc60 mc 15.1 13.5 10.6 2.5 1.0 5.4 - p p p p p p

mc61 mc 15.6 13.9 11.0 2.7 1.1 5.3 - p p p p p p

mc62 mc 16 14.1 11.3 2.9 1.1 5.5 - p p p p p p

mc63 mc 16.1 14.1 11.2 3.0 1.1 5.4 - p p p p p p

mc64 mc 16.5 14.5 11.3 2.9 1.1 6.5 - p p p p p p

mc65 mc 17.1 14.8 11.8 3.1 1.2 6.2 - p p p p p p

mc66 mc 17.3 14.7 11.9 3.0 1.2 6.3 - p p p p p p

mc67 mc 20.8 17.5 13.8 4.1 1.3 6.4 - p p p p p p

mc68 mc 21 17.7 13.7 4.4 1.5 7.8 - p p p p p p

mr1 mr 10 9.6 7.8 1.3 0.5 2.7 na p p p p p p

mr2 mr 10 9.6 7.9 1.3 0.6 2.6 na p p p p p p

mr3 mr 10.05 9.7 8.0 1.3 0.6 2.4 na p p p p p p

mr4 mr 10.3 10.0 8.1 1.2 0.5 2.7 na p p p p p p

mr5 mr 10.4 10.1 8.2 1.3 0.5 2.8 na p p p p p p

mr6 mr 10.7 10.4 8.6 1.4 0.5 2.9 na a p p p p p

mr7 mr 10.7 10.3 8.5 1.4 0.6 2.8 na a p p p p p

mr8 mr 10.8 10.4 8.6 1.4 0.6 2.6 na p p p p p p

mr9 mr 10.8 10.4 8.5 1.4 0.6 2.6 na p p p p p p

mr10 mr 10.9 10.5 8.7 1.4 0.5 2.7 na a p p p p p

mr11 mr 11.2 10.6 8.6 1.6 0.6 3.0 na p p p p u p

mr12 mr 11.3 10.8 8.6 1.5 0.6 3.0 na p p p p p p

mr13 mr 11.3 10.8 8.6 1.6 0.6 2.9 na p p p p p p

mr14 mr 11.6 11.1 8.9 1.6 0.6 3.0 na p p p p p p

mr15 mr 11.6 11.0 8.9 1.7 0.6 3.1 na p p p p p p

mr16 mr 11.6 11.0 8.9 1.7 0.6 3.1 na p p p p u p


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mr17 mr 11.7 11.2 8.9 1.6 0.7 3.1 na p p p p p p

mr18 mr 11.7 11.2 8.9 1.7 0.7 3.0 na p p p p u p

mr19 mr 11.9 11.5 9.1 1.7 0.7 3.1 na p p p p p p

mr20 mr 11.9 11.3 9.0 1.6 0.7 3.2 na p p p p p p

mr21 mr 11.9 11.4 9.2 1.7 0.7 3.1 na p p p p p p

mr22 mr 11.9 11.4 8.9 1.7 0.7 3.1 na p p p p p p

mr23 mr 12 11.4 9.1 2.1 0.8 3.5 na p p p p p p

mr24 mr 12.1 11.4 9.1 1.7 0.7 3.1 na p p p p p p

mr25 mr 12.2 11.5 9.0 1.7 0.8 3.3 na p p p p p p

mr26 mr 12.2 11.7 9.0 1.8 0.8 3.3 na p p p p p p

mr27 mr 12.2 11.5 9.1 1.8 0.7 3.4 na p p p p p p

mr28 mr 12.2 11.5 9.0 2.1 0.8 3.6 na p p p p p p

mr29 mr 12.2 11.6 9.2 2.1 0.8 3.5 na p p p p p p

mr30 mr 12.3 11.7 9.2 1.8 0.8 3.5 na p p p p p p

mr31 mr 12.4 11.8 9.4 1.7 0.8 3.3 na p p p p p p

mr32 mr 12.5 11.8 9.4 2.1 0.7 3.7 na p p p p p p

mr33 mr 12.6 11.9 9.6 1.8 0.7 3.5 na p p p p p p

mr34 mr 13 12.2 9.1 2.2 0.8 4.2 na p p p p p p

mr35 mr 13 12.2 9.3 2.1 0.9 4.0 na p p p p p p

mr36 mr 13.1 12.4 9.6 2.0 0.8 3.7 na p p p p p p

mr37 mr 13.3 12.8 9.6 2.0 0.8 4.3 na p p p p p p

mr38 mr 13.3 12.6 9.8 2.2 0.8 4.0 na p p p p p p

mr39 mr 13.3 12.6 9.4 2.1 0.9 4.2 na p p p p p p

mr40 mr 13.4 12.6 9.2 2.0 0.8 4.0 na p p p p p p

mr41 mr 13.6 13.1 9.8 2.1 0.8 4.4 na p p p p p p

mr42 mr 13.7 13.1 9.6 2.2 0.9 4.2 na p p p p p p

mr43 mr 13.9 13.0 9.8 2.1 0.9 4.0 na p p p p p p

mr44 mr 14 13.0 9.7 2.3 1.0 5.2 na p p p p p p

mr45 mr 14.1 12.6 9.5 2.6 1.0 5.2 na p p p p p p

mr46 mr 14.3 13.6 10.1 2.3 1.0 4.5 na p p p p p p

mr47 mr 14.3 12.8 9.7 2.5 1.0 5.3 na p p p p p p

mr48 mr 14.4 13.6 10.3 2.4 0.9 4.6 na p p p p p p

mr49 mr 14.4 13.7 10.1 2.3 0.9 4.4 na p p p p p p

mr50 mr 14.7 13.7 10.3 2.3 0.9 5.6 na p p p p p p


77


mr51 mr 14.8 12.8 10.0 2.8 1.0 5.5 na p p p p p p

mr52 mr 14.9 13.3 10.1 2.6 1.0 5.5 na p p p p p p

mr53 mr 14.9 13.4 10.1 2.6 1.0 5.4 na p p p p p p

mr54 mr 15 13.3 10.1 2.8 1.0 5.6 2.0 p p p p p p

mr55 mr 15 12.9 9.9 2.7 1.0 5.7 2.2 p p p p p p

mr56 mr 15.2 13.4 10.2 2.7 1.1 5.8 2.0 p p p p p p

mr57 mr 15.3 13.4 10.3 2.7 1.1 5.7 2.0 p p p p p p

mr58 mr 15.4 13.2 10.1 2.8 1.1 5.9 2.1 p p p p p p

mr59 mr 15.5 13.5 10.2 3.0 1.1 5.6 2.3 p p p p p p

mr60 mr 15.5 13.7 10.5 2.7 1.0 6.0 2.2 p p p p p p

mr61 mr 15.7 13.5 10.3 2.8 1.1 5.8 2.2 p p p p p p

mr62 mr 15.7 13.9 10.6 2.9 1.1 5.6 2.3 p p p p p p

mr63 mr 15.7 13.9 10.6 2.7 1.0 6.1 2.2 p p p p p p

mr64 mr 16.3 14.3 11.0 3.3 1.2 6.3 2.5 p p p p p p

mr65 mr 16.3 13.9 10.8 3.0 1.1 6.0 2.3 p p p p p p

mr66 mr 16.3 14.2 10.8 3.2 1.2 6.0 2.7 p p p p p p

mr67 mr 16.4 14.2 10.6 3.1 1.2 6.1 2.5 p p p p p p

mr68 mr 16.5 14.3 10.8 3.2 1.2 6.3 2.5 p p p p p p

mr69 mr 16.7 14.3 11.0 3.3 1.2 6.1 2.6 p p p p p p

mr70 mr 16.7 14.4 11.0 3.2 1.2 6.4 2.3 p p p p p p

mr71 mr 16.7 14.3 10.8 3.2 1.2 6.3 2.5 p p p p p p

mr72 mr 16.8 14.4 11.1 3.2 1.2 6.3 2.5 p p p p p p

mr73 mr 16.8 14.7 11.0 3.2 1.2 6.3 2.5 p p p p p p

mr74 mr 18.9 15.8 11.9 3.8 1.3 6.8 3.0 p p p p p p

mr75 mr 18.9 15.8 11.6 3.9 1.3 7.0 3.1 p p p p p p

mr76 mr 19.2 15.8 12.0 4.0 1.4 7.1 3.2 p p p p p p

mr77 mr 19.2 16.0 11.7 4.0 1.3 7.0 3.3 p p p p p p

mr78 mr 19.4 16.3 11.9 3.9 1.4 7.1 3.4 p p p p p p

mr79 mr 19.6 16.5 12.1 3.9 1.3 7.4 3.3 p p p p p p

mr80 mr 20.1 16.6 12.2 3.8 1.5 7.2 3.2 p p p p p p

mr81 mr 22.7 18.9 13.6 4.2 1.7 8.1 4.0 p p p p p p


78

Table E.1 — continued from previous pageSample # Species Body pigment Pectoral fins Caudal fin Ventral finfold Eye pigment II Head pigment Myosepta pigment Peduncle pigment

mc1 mc a u u a a a u a



mc4 mc p a a a p a a a





mc9 mc p a p a p p p p




mc13 mc p p p p a p p p






















79





































80



mr1 mr a u u a a a u a





mr6 mr p u u a a a u a





mr11 mr p p p p a p p p


mr13 mr p p p p a p p a


mr15 mr p p p p p p p p

mr16 mr p p p a a p p p

mr17 mr p p p a p p p a


mr19 mr p a p p a p p p


mr21 mr p a a p a p p a














81





































82

















83

Table E.1 — continued from previous pageSample # Species Yolk depth Branchiostegals Opercular flaps Caudal fin II Caudal fin III Ventral finfold II Yolk sac pigment Peduncle pigment II

mc1 mc a a a a a a a a








mc9 mc p a a a a a a a



mc12 mc p a a a a p a a

mc13 mc a p p p p p a p














mc27 mc a p p p p p p a








84





































85



mr1 mr a a a a a a a a






















mr23 mr a a p p p p a a

mr24 mr a p p a a a a a

mr25 mr a p p p p p p a

mr26 mr a a a a p a p a

mr27 mr a p a p p p p a

mr28 mr a p p p a p a a

mr29 mr a a p p p a a a

mr30 mr a p p p a p p a

mr31 mr a p p p a a a a

mr32 mr a a p p p p p a

mr33 mr a p p p p p a a


86





































87

















88

Table E.1 — continued from previous pageSample # Species Yolk depth II Nares Digestive tract Caudal fin IV Urostyle Dorsal fin Ventral finfold III Dorsum pigment

mc1 mc a a a u u a a a













mc14 mc a a a a a a a p

mc15 mc a a a a a p a a



mc18 mc a a a a a a a p

mc19 mc a a a p a a a p

mc20 mc a a a p a a p a





mc25 mc a a a p a a a a

mc26 mc a a a p a a a a

mc27 mc p p p p p p p a


mc29 mc p p p p p p p p






89



mc35 mc p p p p p p a a




mc39 mc p p p p p p a p


mc41 mc p p p p p p a a




























90



mr1 mr a a a u u a a a


































91


mr34 mr p p a p p p a p

mr35 mr p p p p p p p a

mr36 mr a p a p p p a p

mr37 mr a p a p p a p p

mr38 mr p p a p p p p p

mr39 mr a p a p p p p p




mr43 mr p p a p p p p p






mr49 mr p p p p p p a p




















92

















93

Table E.1 — continued from previous pageSample # Species Chin pigment Yolk sac Mouth position Pelvic fins Dorsal fin margins Dorsal finfold Anal fin Arrow-shaped pigment

mc1 mc p a u a u u a a


























mc27 mc p a a a a a p a

mc28 mc p a a a a a a p

mc29 mc p a a a a a p p






94













mc45 mc p p a a a a p p




mc49 mc p a a a a a p a


mc51 mc a p a a a a p p









mc60 mc a p p p p p p p









95



mr1 mr p a u a u u a a


































96













mr45 mr a p p p p p a p

mr46 mr a a a p p p a a


mr48 mr a p a p p p a a

mr49 mr a a p p p p p p

mr50 mr a p p p p p p p



















97

















98

Table E.1 — continued from previous pageSample # Species Gut melanophores Jaw pigment Gill arch pigment Mouth position II Anal fin II Pelvic flaps Dorsal finfold II Ventral finfold IV

mc1 mc a a a a a a u u


































99




























mc60 mc a p a a a a a a

mc61 mc a p p a a a a a








100



mr1 mr a a a a a a u u


































101












mr44 mr a p a a a a a a

mr45 mr p p a a a a a a
























102

















103

Table E.1 — continued from previous pageSample # Species Operculum length Pelvic fins Lip pigment Snout pigment Jaw pigment II Head profile Dorsal fin profile Anal fin III

mc1 mc u u u u u a u u


































104






























mc62 mc a p a a u a u u



mc65 mc p p a a a a a a

mc66 mc p p a a a a a a

mc67 mc p p a a p a a p


105


mc68 mc p p a a p a a p

mr1 mr u u u u u a u u


































106






















mr54 mr u a u u u a u u










mr64 mr p p a a u a u u



mr67 mr a p a a u a u u


107








mr74 mr p p a a p p p p

mr75 mr p p a a a p p p








108

Table E.1 — continued from previous pageSample # Species Pelvic fins II Dorsal finfold III Squamation Scale pigment Dorsum pigment II

mc1 mc u u a u u

mc2 mc u u a u u

mc3 mc u u a u u

mc4 mc u u a u u

mc5 mc u u a u u

mc6 mc u u a u u

mc7 mc u u a u u

mc8 mc u u a u u

mc9 mc u u a u u

mc10 mc u u a u u

mc11 mc u u a u u

mc12 mc u u a u u

mc13 mc u u a u u

mc14 mc u u a u u

mc15 mc u u a u u

mc16 mc u u a u u

mc17 mc u u a u u

mc18 mc u u a u u

mc19 mc u u a u u

mc20 mc u u a u u

mc21 mc u u a u u

mc22 mc u u a u u

mc23 mc u u a u u

mc24 mc u u a u u

mc25 mc u u a u u

mc26 mc u u a u u

mc27 mc u u a u u

mc28 mc u u a u u

mc29 mc u u a u u

mc30 mc u u a u u

mc31 mc u u a u u

mc32 mc u u a u u

mc33 mc u u a u u


109


mc34 mc u u a u u

mc35 mc u u a u u

mc36 mc u u a u u

mc37 mc u u a u u

mc38 mc u u a u u

mc39 mc u u a u u

mc40 mc u u a u u

mc41 mc u u a u u

mc42 mc u u a u u

mc43 mc u u a u u

mc44 mc u u a u u

mc45 mc u u a u u

mc46 mc u u a u u

mc47 mc u u a u u

mc48 mc u u a u u

mc49 mc u u a u u

mc50 mc u u a u u

mc51 mc u u a u u

mc52 mc u u a u u

mc53 mc u u a u u

mc54 mc u u a u u

mc55 mc u u a u u

mc56 mc u u a u u

mc57 mc u u a u u

mc58 mc u u a u u

mc59 mc u u a u u

mc60 mc u u a u u

mc61 mc u u a u u

mc62 mc u u a u u

mc63 mc u u a u u

mc64 mc u u a u u

mc65 mc a p a u u

mc66 mc p a a u u

mc67 mc a p a p p


110


mc68 mc p a a a p

mr1 mr u u a a u

mr2 mr u u a a u

mr3 mr u u a a u

mr4 mr u u a a u

mr5 mr u u a a u

mr6 mr u u a a u

mr7 mr u u a a u

mr8 mr u u a a u

mr9 mr u u a a u

mr10 mr u u a a u

mr11 mr u u a a u

mr12 mr u u a a u

mr13 mr u u a a u

mr14 mr u u a a u

mr15 mr u u a a u

mr16 mr u u a a u

mr17 mr u u a a u

mr18 mr u u a a u

mr19 mr u u a a u

mr20 mr u u a a u

mr21 mr u u a a u

mr22 mr u u a a u

mr23 mr u u a a u

mr24 mr u u a a u

mr25 mr u u a a u

mr26 mr u u a a u

mr27 mr u u a a u

mr28 mr u u a a u

mr29 mr u u a a u

mr30 mr u u a a u

mr31 mr u u a a u

mr32 mr u u a a u

mr33 mr u u a a u


111


mr34 mr u u a a u

mr35 mr u u a a u

mr36 mr u u a a u

mr37 mr u u a a u

mr38 mr u u a a u

mr39 mr u u a a u

mr40 mr u u a a u

mr41 mr u u a a u

mr42 mr u u a a u

mr43 mr u u a a u

mr44 mr u u a a u

mr45 mr u u a a u

mr46 mr u u a a u

mr47 mr u u a a u

mr48 mr u u a a u

mr49 mr u u a a u

mr50 mr u u a a u

mr51 mr u u a a u

mr52 mr u u a a u

mr53 mr u u a a u

mr54 mr u u a a u

mr55 mr u u a a u

mr56 mr u u a a u

mr57 mr u u a a u

mr58 mr u u a a u

mr59 mr u u a a u

mr60 mr u u a a u

mr61 mr u u a a u

mr62 mr u u a a u

mr63 mr u u a a u

mr64 mr u u a a u

mr65 mr u u a a u

mr66 mr u u a a u

mr67 mr u u a a u


112


mr68 mr u u a a u

mr69 mr u u a a u

mr70 mr u u a a u

mr71 mr u u a a u

mr72 mr u u a a u

mr73 mr u u a a u

mr74 mr p p a a u

mr75 mr p p a a u

mr76 mr p p a a u

mr77 mr p p a a u

mr78 mr p p a a u

mr79 mr p p a a u

mr80 mr p p p p p

mr81 mr p p p p p


river, georgia j. stuart carlton abstract

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