nucleotide sequence data confirm diagnosis and local...

Nucleotide sequence data confirm diagnosis and localendemism of variable morphospecies of Andeanastroblepid catfishes (Siluriformes: Astroblepidae)

SCOTT A. SCHAEFER1*, PROSANTA CHAKRABARTY2, ANTHONY J. GENEVA3,4 andMARK H. SABAJ PÉREZ4

1American Museum of Natural History, Division of Vertebrate Zoology, Central Park West at 79thSt.,New York, NY 10024, USA2Museum of Natural Science, Louisiana State University, 119 Foster Hall, Baton Rouge, LA 70803,USA3Department of Biology, University of Rochester, Rochester, NY 14627, USA4Academy of Natural Sciences, Department of Ichthyology, 1900 Ben Franklin Pkwy., Philadelphia,PA 19103, USA

Received 24 November 2009; accepted for publication 27 April 2010

Phylogenetic analysis based on nuclear and mitochondrial DNA sequences was used to test the validity ofmorphospecies of catfishes of the family Astroblepidae inhabiting the southern-most limit of their Andeandistribution in the upper Ucayali and upper Madre de Dios river basins. Population samples of morphospeciesdesignated a priori on the basis of morphological features were further diagnosed by the presence of unique andunreversed molecular synapomorphies, thereby confirming species validity for seven of nine cases. Although eachare distinguished by unique combinations of morphological features, two morphospecies (designated F and H)cannot be diagnosed on the basis of apomorphic changes in molecular sequence that did not also occur in otherastroblepid morphospecies or outgroup taxa. Further, one morphospecies (species G) was recovered as nestedwithin the assemblage of populations sampled from morphospecies F, whose morphological diagnosis does notinvolve unique or apomorphic characters. In contrast, the absence of corroborating molecular apomorphies forspecies H, otherwise recognized by distinctive and uniquely derived morphological characters, suggests a historyof rapid divergence and insufficient time for fixation of genetic differences. Species sharing syntopic distributionswere not recovered as sister groups, and in some cases species distributed in adjacent river drainage basins werenot more closely related to one another than to species distributed in more distant drainages. Three independentinstances were observed of sister-group relationships involving species distributed in both the Apurimac andUrubamba rivers (Ucayali drainage). These observations combine to suggest that the current distribution ofastroblepid species in the southern region may have arisen via a complex history involving both divergence betweenand dispersal amongst drainage basins that is probably repeated numerous times throughout the Andeandistribution of the group.

© 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011.doi: 10.1111/j.1096-3642.2010.00673.x

ADDITIONAL KEYWORDS: Andes – biogeography – evolution – ichthyology – South America – speciesconcepts – taxonomy.

INTRODUCTION

Astroblepid catfishes represent a distinctive assem-blage of species that live at moderate to high eleva-

tions in freshwaters of the tropical Andes. Theirdistribution extends from Panama to Bolivia andacross nearly 28° of latitude. Within that range,astroblepids occur in all of the major river drainagesystems of the Pacific, Caribbean, and Amazon-Orinoco basins. Most species are of moderate to small*Corresponding author. E-mail: [email protected]

Zoological Journal of the Linnean Society, 2011. With 3 figures

© 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011 1

size, typically less than 0.10 m in length, but occa-sionally reach about 0.30 m as adults. Astroblepids,commonly known as climbing catfishes, are easilyrecognized by their expanded fleshy oral disk andthickened, highly mobile pelvic fins, with which theyadhere to the substratum and locomote in the high-gradient, rapidly flowing streams that characterizetheir montane habitats. In contrast to their sistergroup, the mega-diverse catfishes of the family Lori-cariidae (96 genera, 716 species; Ferraris, 2007),which are widespread in lowland rivers throughoutthe Neotropics, astroblepids are presently classified ina single genus (Astroblepus) and 54 species that arestrictly Andean in distribution (Schaefer, 2003). Thereis no fossil record. With few exceptions, most speciesof Astroblepus have restricted geographical distribu-tions, being limited to portions of single river drain-age basins at elevations above 1000 m (Schaefer,2003). In contrast, amongst the more species-richgenera of the Loricariidae having been the subject ofrecent taxonomic revisions involving comprehensiveexamination of material (e.g. Panaque – Schaefer &Stewart, 1993; Otocinclus – Schaefer, 1997; Oxyropsis– Aquino & Schaefer, 2002), a much larger proportionof the specific diversity is represented by specieshaving broader geographical distributions (Ferraris,2003; Fisch-Muller, 2003; Weber, 2003). The dispari-ties in taxonomic diversity and distribution and theestimated age of divergence between astroblepids andtheir sister group (approx. 90 Mya; Sullivan, Lund-berg & Hardman, 2006) relative to the much youngerage (approx. 10 Myr) for higher elevations (above2 km) in the Andes (Gregory-Wodzicki, 2000; Garzi-one et al., 2008) and rapid rates of recent speciesdiversification observed for some plants at elevation(Hughes & Eastwood, 2006), pose several interestingquestions regarding the timing of family-level diver-gence and rates of evolution within Neotropical cat-fishes. Furthermore, astroblepids themselves, as animportant component of the poorly known and dep-auperate Andean fish fauna, are potentially impor-tant biotic indicators of the health of criticallyimportant source headwaters of the major rivers ofthe Neotropics.

Knowledge of the taxonomy, diversity, and ecologyof astroblepid catfishes is rudimentary because therehave been no synthetic revisionary studies of astrob-lepids since the monographic work of Regan (1904).Most of the species are known only from their originaldescriptions and all but four of the 54 nominal specieswere described before 1950. At present, it is difficultto distinguish species because most are defined onlyby single-character contrasts or by overlapping andnon-unique combinations of external features thatdisplay high levels of inter- and intraspecific varia-tion. During the course of a taxonomic revision of the

family conducted by the first author, it became appar-ent that traits used in defining the morphologicallimits between astroblepid species, most notably, bodyshape, fin size and configuration, and pigmentationpattern, are confounded by variation on severallevels. For example, observed patterns of morphologi-cal variation appear to be the result of complex con-tributions from multiple intrinsic and extrinsicsources, such as ontogeny, sexual dimorphism, andgeographical variation. Pigmentation patterns on thehead and trunk, in particular, are highly variablewithin and amongst species (Fig. 1) to an extent thatapplication of independent sources of data are neces-sary for evaluating concepts of astroblepid mor-phospecies defined in part by coloration pattern.

Application of DNA-based approaches to taxonomicquestions (Hebert et al., 2003) can be useful in thesecircumstances because the introduction of molecularcriteria can supplement classic morphological andbehavioural criteria in judging species boundariesand recognizing hitherto undiscovered diversity(DeSalle, Egan & Siddal, 2005). Population geneticsapproaches are often most appropriate in cases whereputative species are highly polymorphic, suggestingthat traits may have not become fixed and where geneflow via migration and hybridization operate tooppose segregation and differentiation. As theseapproaches can be demanding and time consuming,we are most interested in using simplified proceduresfor assessing species status that avoid makingassumptions about divergence threshold (Hebertet al., 2003), divergence time (Pons et al., 2006), popu-lation size or number of generations required toachieve reciprocal monophyly (Hudson & Coyne,2002), or other attributes of astroblepid populationsthat are unknown at present. Following DeSalle et al.(2005), we reject species delimitation on the basis ofdistance-based methods (e.g. based on amount ordegree of divergence), as opposed to character-basedapproaches using DNA sequence data, because onlythe latter are compatible with current taxonomicprinciples and objective hypothesis tests of speciesdiagnosis.

The goals of this study were to test a priori mor-phospecies designations of astroblepid catfishes usingmultigene nucleotide sequence data. We applied thephylogenetic species concept (Nixon & Wheeler, 1990)and used the criterion of autapomorphy (unique,unreversed derived change in molecular sequence;DeSalle et al., 2005) in testing the validity of putativespecies. A phylogenetic analysis of the molecular dataset was used to infer the optimization of molecularcharacters on the tree, although, following DeSalleet al. (2005), we did not utilize the pattern ofrelationships amongst morphospecies in the testof species validity because species need not be

2 S. A. SCHAEFER ET AL.

© 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011

Figure 1. Variation in pigmentation in Astroblepus morphospecies A–I. A, morphospecies A, ANSP (Academy of NaturalSciences of Philadelphia) 180586 (4793), 51.6 mm standard length (SL), Araza River. B, morphospecies B, ANSP 180587(4779), 75 mm SL, Araza River. C, morphospecies B, ANSP 180582 (4801), 80.4 mm SL, Araza drainage (Dr.) D,morphospecies B, ANSP 180582 (4800), 54.5 mm SL, Araza Dr. E, morphospecies C, ANSP 180581 (4805), 27.2 mm SL,Araza Dr. F, morphospecies C, ANSP 180586 (4794), 58 mm SL, Araza River. G, morphospecies D, ANSP 180599 (4822),51.7 mm SL, Urubamba Dr. H, morphospecies D, ANSP 180602 (4499), 85 mm SL, Urubamba Dr. I, morphospecies H,ANSP 180618 (4423), 46.3 mm SL, Apurimac Dr. J, morphospecies H, ANSP 180616 (4436), 79.2 mm SL, Apurimac Dr.K, morphospecies E, ANSP 180595 (4785), 61.3 mm SL, Urubamba Dr. L, morphospecies E, ANSP 180605 (4490),110.5 mm SL, Apurimac Dr. M, morphospecies F, ANSP 180606 (4487), 75.7 mm SL, Apurimac Dr. N, morphospecies F,ANSP 180601 (4759), 52.6 mm SL, Urubamba Dr. O, morphospecies G, ANSP 180588 (4787), 59.5 mm SL, Urubamba Dr.P, morphospecies I, ANSP 180607 (4477), 39.4 mm SL, Apurimac Dr. Photo in (A) by S. A. S.; photos in (B–P) by M. H.S. P.

MOLECULAR DIAGNOSIS OF ASTROBLEPID SPECIES 3


monophyletic (type-C monophyly of Rieppel, 2009). Forreasons of efficacy and feasibility, we applied this testto the astroblepid species of southern Peru, the south-ern limit of the distribution of the family and a keyregion for understanding the historical and ecologicalfactors that determine astroblepid distribution. Thestudy region is physically and ecologically complex andincludes a diversity of landforms and ecoregions,where biotic assemblages are greatly impacted byinteractions amongst precipitation, temperature, andtopography that vary greatly on regional scales(Killeen et al., 2007). These factors combine to define atransition zone in the pattern of distribution andendemism between the south-central and southernAndean biotas (Sarmiento, 1975; Kessler, 2002; López,2003). Diversity and endemism of astroblepid speciesin this region is high, with eight nominal and 13morphospecies distributed in the Madre de Dios, Beni,Ucayali, and Titicaca watersheds.

MATERIAL AND METHODSSTUDY REGION AND SPECIMENS EXAMINED

The study region was defined as the freshwaters ofthe central portion of the Central Andes (Gregory-Wodzicki, 2000) of southern Peru and northernBolivia between 10° and 18°S latitude (Fig. 2). The

study region encompasses the major Andean headwa-ter tributaries of the Amazon lowlands, including theinter-Andean upper Ucayali River and its southerntributaries (Apurimac and Urubamba), and theMadre de Dios and Beni/Madeira rivers of theAmazon fore slope to the south-east. Withinthe Ucayali drainage, the drainages of the Apurimacand Mantaro rivers on the west are separated fromthose of the Urubamba River on the east by theCordillera Vilcabamba, whereas the combinedUcayali drainages are separated from the Amazonfore slope drainages by the Vilcanota, Carabaya, andApolobamba ranges. Although astroblepids also occurin both the Pacific slope and isolated Titicaca drain-ages, there are extremely few verified locality recordsfor astroblepid species in these portions of the studyregion and therefore these taxa were excluded.

Specimens examined were assembled from themajor international ichthyological collections withholdings of Andean fishes (Appendix S1; codes forinstitutional repositories are as listed at http://www.asih.org/node/204). Veracity of locality dataassociated with the specimen records was checkedagainst multiple gazetteers and literature sources.Locality records were geocoded and input to a geo-graphical information system (ArcView, v. 9.3) andvisualized on a three arc-sec digital elevation model

Figure 2. Distribution of astroblepid morphospecies and study region. Circled letters correspond with the morphospeciesdesignations (Table 1) and may represent more than one lot or collection locality.



(DEM) obtained from the USGS/NASA Shuttle RadarTopography Mission (Jarvis et al., 2006). Additionalspecimens were obtained by fieldwork in 2004; theselocalities were coded in the field by a global position-ing system.

CRITERIA FOR DEFINING AND TESTING

MORPHOSPECIES

Fixed and discrete states of homologous features wererecorded from a variety of external morphologicalsystems and used to assign astroblepid specimens tophenetic morphospecies. Specimens were treated aspopulation samples and morphospecies were recog-nized by application of the diagnosability criterion(Nixon & Wheeler, 1990): those populations sharingthe smallest mutually exclusive set of unique featuresand/or unique combinations of features. Geographicalorigin of specimens was ignored when assigningspecimens to morphospecies. We used the phyloge-netic species concept (Mayden, 1997; de Queiroz,2007) in the test of morphospecies validity by appli-cation of the criterion of autapomorphy (Rosen, 1979;Wheeler & Platnick, 2000). Validity of morphospeciesdefined a priori on the basis of phenetic criteria wasrejected when not further corroborated by the pres-ence of unique and unreversed changes in the inde-pendent multigene molecular sequence data.

MOLECULAR DATA AND PHYLOGENETIC ANALYSES

A total of 37 samples representing nine astroblepidmorphospecies collected from 24 field sites was usedin this study (Table 1). Tissues (fin clips, liver, ormuscle) were sampled and preserved in 95% ethanolprior to specimen fixation in 10% formalin, or subse-quently transferred to 95% ethanol (for long-termstorage at -80 °C) from specimens field-preserved in70% ethanol. Additional voucher specimens were fixedin formalin and transferred to 70% ethanol. Addition-ally, six samples of astroblepid species collected fromlocalities external to the study area were included,along with four species of Loricariidae as outgroups.Tissue, GenBank, and voucher specimen numbers forall taxa examined are listed in Table 1.

We obtained a total of 3217 base pairs (bp) of DNAsequence from the following genes: recombinationactivating gene 1 (Rag-1; 1355 bp), cytochrome coxidase subunit I (COI; 658 bp), cytochrome b (cytb;629 bp), and 16S rRNA (16S; 575 bp). Total DNA wasextracted using a Qiagen DNEasy tissue extractionkit following the manufacturer’s protocol. The Rag-1fragment was amplified and sequenced using theprimers F74, R1333, F354, and R798 as specified inSullivan et al. (2006: Table 1). The COI fragmentwas amplified and sequenced using the primersLCO1490 5′-GGTCAACAAATCATAAAGATATTGG-3′

and HCO2198 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ (Folmer et al., 1994) or Pros1Fwd 5′-TTCTCGACTAATCACAAAGACATYGG-3′ and Pros2Rev5′-TCAAARAAGGTTGTGTTAGGTTYC-3′ (‘COIfor’and ‘COIrev’ from Chakrabarty, 2006). The cytb frag-ment was amplified and sequenced using theprimers ICytb-F1 5′-TTCCTTYCACCCCTATTTCT-3′and ICytb-R1 5′-CTGGGGTGAAGTTTTCTGGG-3′(Hardman & Page, 2003). The 16S fragment wasamplified and sequenced using the primers 16Sar-L 5′-CGCCTGTTTATCAAAAACAT-3′and 16S br-H5′-CCGGTCTGAACTCAGATCACGT- 3′ (Kocher et al.,1989; Palumbi, 1996). Double-stranded amplificationproducts were desalted and concentrated usingAMPure (Agencourt Biosciences Corp.) or ExoSAO-IT(USB Corp.). Both strands of the purified PCR frag-ments were used as templates and directly cyclesequenced using the original amplification primersand an ABI Prism Big Dye Terminator Reaction Kit(versions 1.1, 3.1). The sequencing reactions werecleaned and desalted using cleanSEQ (Agencourt Bio-sciences Corp.) or BigDye X-Terminator (Applied Bio-systems Corp.). The sequencing reactions wereelectrophoresed on an ABI 3730xl automated DNAsequencer. Contigs were built in SEQUENCHERversion 4.8 (Gene Codes, Ann Arbor, MI, USA) usingDNA sequences from the complementary heavy andlight strands. Sequences were edited inSEQUENCHER and BIOEDIT (Hall, 1999), alignedusing ClustalX (Larkin et al., 2007), and modified byeye. All novel sequences have been deposited inGenBank under accession numbers HM048988-49165(Table 1).

A total of 3217 aligned bp from the four genefragments was analysed. Our multigene data set rep-resents an approximate 50 : 50 assemblage of bpdrawn from mitochondrial and nuclear markers.Although data derived from mitochondrial genes canbe readily obtained and have proven to be effective indiverse studies of fishes (Farias et al., 1999; Miyaet al., 2003), these data are less reliable than nucleargene markers under situations involving rapid diver-gence and incomplete lineage sorting of mtDNA hap-lotypes over relatively short branches, and horizontaltransfer of genes across populations (Hudson &Coyne, 2002). Given the absence of pre-existing infor-mation on the performance of genomic markers forastroblepid catfishes and lack of insight on theirpopulation biology, we therefore adopted a conserva-tive approach and compared the phylogenetic signalsprovided by the nuclear and mitochondrial data setsboth separately and combined under a total-evidenceapproach (Eernisse & Kluge, 1993; Nixon & Carpen-ter, 1996; Frost et al., 2001) using both maximumlikelihood (ML) and parsimony (MP) optimality crite-ria. ML analyses and bootstrap calculations were



Tab

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4782

Ast

robl

epu

sF

1805

94H

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9142

HM

0490

99H

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9056

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0490

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ruba

mba

,R

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nat

ili

nea

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uen

cew

ith

R.

Uru

bam

ba48

39A

stro

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us

F18

0594

HM

0491

59H

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9115

HM

0490

73H

M04

9027

Uru

bam

ba,

R.

Yan

atil

in

ear

con

flu

ence

wit

hR

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ruba

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4787

Ast

robl

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sG

1805

88H

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9144

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0491

00H

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9058

HM

0490

12U

ruba

mba

,R

.M

apit

un

ari

Nof

Kit

eni

4750

Ast

robl

epu

sG

1805

88H

M04

9138

HM

0490

95H

M04

9052

HM

0490

06U

ruba

mba

,R

.M

apit

un

ari

Nof

Kit

eni

4470

Ast

robl

epu

sH

1806

09H

M04

9129

HM

0490

88H

M04

9043

HM

0489

97A

puri

mac

,R

.C

him

bao

ups

trea

mof

An

dah

uay

las

4416

Ast

robl

epu

sH

1806

18H

M04

9123

HM

0490

83H

M04

9037

HM

0489

91A

puri

mac

,R

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ucr

en

ear

tow

nof

Lu

cre,

NE

ofC

olca

bam

ba44

23A

stro

blep

us

H18

0618

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0491

24H

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9084

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0490

38H

M04

8992

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rim

ac,

R.

Lu

cre

nea

rto

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ofL

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Col

caba

mba

4477

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robl

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1806

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9130

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0490

89H

M04

9044

HM

0489

98A

puri

mac

,R

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ampa

s,W

ofC

hin

cher

os66

41A

stro

blep

us

sp18

8865

HM

0491

60H

M04

9117

HM

0490

74H

M04

9029

Mag

dale

na,

R.

San

Fra

nci

sco,

Cu

ndi

nam

arca

,C

olom

bia

P60

58A

stro

blep

us

spA

UM

4655

9H

M04

9164

HM

0491

21H

M04

9078

HM

0490

33M

aran

on,

Q.

Sia

sme,

Con

dorc

anqu

i,A

maz

onas

,P

eru

P60

59A

stro

blep

us

spA

UM

4655

9H

M04

9165

HM

0491

22H

M04

9079

HM

0490

34M

aran

on,

Q.

Sia

sme,

Con

dorc

anqu

i,A

maz

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,P

eru

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41A

stro

blep

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spA

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4653

6H

M04

9162

HM

0491

19H

M04

9076

HM

0490

31M

aran

on,

R.

Alm

endr

o,C

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iaco

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maz

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,P

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42A

stro

blep

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spA

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4653

6H

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9163

HM

0491

20H

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9077

HM

0490

32M

aran

on,

R.

Alm

endr

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hir

iaco

,A

maz

onas

,P

eru

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20A

stro

blep

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spA

UM

4652

2H

M04

9161

HM

0491

18H

M04

9075

HM

0490

30M

aran

on,

R.

Hu

anca

bam

ba,

Piu

ra-C

ajam

arca

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eru

–L

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arcu

sm

ult

irad

iatu

sIN

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5458

5D

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2605

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HM

0490

28O

rin

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Mar

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Por

tugu

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Ven

ezu

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arlo

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reri

1827

79D

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2578

HM

0490

82H

M04

9036

HM

0489

90A

maz

on,

R.

Sol

imõe

s,A

maz

onas

,B

razi

l41

81L

oric

aria

sim

illi

ma

1804

98D

Q49

2607

HM

0490

80–

HM

0489

88M

adre

deD

ios,

R.

Inam

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uzc

o,P

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4182

Lam

onti

chth

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0635

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4926

02H

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9081

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rede

Dio

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SP,

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phia

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east

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nal

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ter

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conducted on individual gene partitions as well as onthe concatenated data set in RaxML 7.0.4 using theCipres Portal v. 1.15 implementing a general timereversible (GTR) + gamma model as recommended(Stamatakis, Hoover & Rougemont, 2008). Partitionswere based on gene fragments and codon position,when applicable. The number of bootstrap replicates(250 Rag-1, cytb; 200 COI, 400 16S; 150 concatenateddata set) was automatically determined during theruns as adequate and rigorous by RaxML for eachdata set. MP analyses were conducted on the concat-enated data set using TNT v. 1.1 (Goloboff, Farris &Nixon, 2008) using traditional heuristic searches, tenrandom taxon addition sequences, tree bisectionreconnection (TBR) with 30 replicates and ten treesper replicate. Indels and substitutions were weightedequally.

RESULTS

Our survey of astroblepid external morphologyresulted in the recognition of nine morphospecies(Fig. 1; species designated A–I, material examinedlisted in Appendix S1). A tenth morphospecies, corre-sponding to the nominal Astroblepus longiceps, wasrecognized as the sole representative of the genus inBolivia, but was excluded from the test of morphospe-cies status because of a lack of tissue samples. Four ofthe nine morphospecies (A, B, C, G) are restricted indistribution to a single drainage basin, with threespecies (A, B, C; Fig. 1) occurring sympatrically atmultiple localities within the Madre de Dios riversystem. The remaining five morphospecies (D, E, F, H,I) each have a wider geographical distribution andoccur in more than one drainage basin within thestudy region (Fig. 2).

For the combined data set of 3217 nucleotides, 1007sites were variable and 766 of these were parsimonyinformative. ML analysis of the concatenatedsequence data run with joint branch length optimiza-tion yielded the highest likelihood score of ln-13710.612764 (Fig. 3). For the partitioned data sets,amongst individual trees (not shown), the best scoreswere ln -2065.012655 (16S), ln –3010.527911 (COI),ln -3635.814303 (cytb), ln -4652.939822 (Rag-1). MPanalyses on the concatenated data set yielded 28equally most-parsimonious trees of length = 2186,consistency index = 0.64, retention index = 0.83. Thestrict consensus amongst these trees yielded a topol-ogy identical to that obtained from the ML analysis interms of recovered species assemblages and relation-ships amongst the morphospecies. Monophyly ofAstroblepidae was strongly supported in all analyses,but the morphospecies of the study region were notrecovered as monophyletic because sample 6020

Astroblepus sp. (Marañon River) nested within theingroup at an identical position amongst the ML andMP trees.

Six of nine astroblepid morphospecies designated apriori on the basis of morphological characteristicswere recovered as monophyletic in all analyses(Fig. 3). Two of nine morphospecies (A, I) were bothrepresented in the phylogenetic analyses by a singlespecimen, and therefore monophyly of these speciescannot be falsified. Morphospecies G was recovered asnested within a monophyletic assemblage that alsoincluded individuals of morphospecies F (Fig. 3).Within the ingroup, most nodes, including thoseindicative of morphospecies monophyly, were wellsupported in the bootstrap analyses (bootstrap pro-portions > 80%). The combined species F+G clade wasrecovered as the sister group to a well-supportedspecies E. Species B and C were each recovered asmonophyletic and placed in a well-supported cladeincluding species E and F+G; that clade sister to onecomposed of species A, I and sample 6020 from theMarañon. Sister species D and H were recovered asthe sister group to the clade inclusive of all othermorphospecies and sample 6020.

Seven of the nine morphospecies were each associ-ated with one or more unique and unreversed bpchanges amongst the molecular sequences examined.These uniquely derived molecular characters, com-bined with the unique morphological features orunique combinations of characters, serve to diagnosethese seven morphospecies (Table 2). Two of the ninemorphospecies (F, H) are not diagnosed by any auta-pomorphic molecular characters, and therefore failour test of species status.

DISCUSSION

Our analysis recovered a monophyletic Astroblepus,but the nine morphospecies of the study region do notrepresent a monophyletic assemblage, exclusive ofspecies from other geographical regions. Despite theoccurrence of unique combinations of morphologicalfeatures useful for the identification of all nine mor-phospecies, our analysis of combined mitochondrialand nuclear gene sequence data sets failed to identifyunique molecular characters for two of the nine mor-phospecies (F and H). Applying the criterion of apo-morphy under the phylogenetic species concept(Wheeler & Platnick, 2000), and in the absence ofcorroboration provided by the molecular data, wewould reject species status for these two morphospe-cies. This outcome is both surprising and illuminatingwith respect to the utility of the morphological fea-tures hypothesized at the outset to define these par-ticular morphospecies.



The phylogenetic analyses uniformly recovered anonmonophyletic species F, because of the fact thatindividuals assigned a priori to species G were recov-ered as nested within the assemblage of population

samples for species F. Although the finding of non-monophyly for species F does not factor into our testof morphospecies status, because species need not betype-C monophyletic (Rieppel, 2009), the absence of

Figure 3. Results of the phylogenetic analysis of astroblepid morphospecies obtained from maximum likelihood analysisof the combined DNA sequence data set. Numerals at nodes represent bootstrap proportions (values less than 50% notshown); stars represent nodes supported by bootstrap values of 80% or greater. Sample numbers correspond withmaterials listed in Table 1. Letters designate morphospecies; shaded boxes denote monophyletic assemblages of popula-tion samples.



Tab

le2.

Mor

phol

ogic

alan

dm

olec

ula

rdi

agn

osis

ofA

stro

blep

us

mor

phos

peci

es

Taxo

nM

orph

olog

yC

OI

(1–6

58)

Cyt

b(6

59–1

287)

16S

(128

8–18

62)

Rag

1(1

863–

3217

)

AB

road

sym

met

rica

lly

bifi

dpr

emax

illa

teet

h,

adip

ose

spin

eab

sen

t,m

axil

lary

barb

eln

otto

post

erio

rli

pm

argi

n

(94:

T),

(277

:T),

(401

:T),

(508

:G),

(571

:C),

(589

:T)

(744

:T),

(812

:A),

(815

:T),

(839

:A),

(932

:G),

(108

3:G

),(1

151:

T),

(116

9:C

),(1

169:

G)

(155

6:T

),(1

574:

T)

(161

7:T

),(1

695:

C)

(201

0:T

),(2

180:

C),

(227

4:A

),(2

416:

A),

(313

3:T

)

BP

rem

axil

lary

teet

hu

nic

usp

id,

adip

ose-

fin

mem

bran

eta

ll,

adip

ose

spin

eab

sen

t,m

axil

lary

barb

elex

ten

ded

beyo

nd

post

erio

rli

pm

argi

n,

tru

nk

mot

tled

(536

:T)

(966

:T),

(104

6:G

)–

(234

5:G

),(2

605:

A)

CM

axil

lary

barb

eln

otto

post

erio

rli

pm

argi

n,

pect

oral

-fin

rays

11–1

2(7

0:G

),(1

93:T

),(5

32:T

),(5

42:T

)(7

85:T

),(7

97:T

),(8

24:T

),(9

99:G

),(1

000:

C),

(128

4:C

)

(140

0:A

),(1

705:

C)

(216

2:A

),(2

595:

A),

(260

5:G

)

DM

axil

lary

barb

elex

ten

ded

beyo

nd

post

erio

rli

pm

argi

n,

pore

soc3

sepa

rate

dfr

omso

c2by

dist

ance

less

than

post

erio

rn

aris

diam

eter

(40:

C),

(430

:G),

(544

:T),

(586

:G)

(763

:T),

(773

:T),

(824

:G),

(833

:G),

(890

:C),

(105

8:G

),(1

133:

T),

(113

8:G

)

(171

5:T

)(2

474:

T),

(258

3:A

),(2

646:

G),

(282

2:T

),(2

996:

A),

(302

8:T

),(3

097:

C),

(312

0:C

)

ED

enta

ryco

vere

dve

ntr

ally

byex

ten

sion

oflo

wer

lip

(299

:A),

(359

:C)

––

(233

2:A

),(3

111:

T)

FA

dipo

sem

embr

ane

not

tru

nca

ted,

con

tin

ued

onto

cau

dal

pedu

ncl

e;ad

ipos

esp

ine

pres

ent;

pmx

teet

hei

ght

tote

n;

max

illa

ryba

rbel

lon

g,re

ach

ing

beyo

nd

post

erio

rm

argi

nof

low

erli

p;de

nta

ryn

otco

vere

dby

exte

nsi

onof

low

erli

p

––

––

GP

rem

axil

lary

and

den

tary

teet

has

ymm

etri

call

ybi

fid;

adip

ose

spin

eab

sen

t

––

–(2

173:

G)

HM

andi

bula

rra

mu

sn

arro

w;

post

erio

rli

pla

min

aw

ide,

extr

emel

yde

ep–

––

–

IA

dipo

sem

embr

ane

abse

nt,

adip

ose

spin

ese

para

te,

elev

ated

,m

axil

lary

barb

elsh

ort,

not

toli

pm

argi

n,

bico

lou

red

pigm

enta

tion

(334

:A),

(550

:T),

(671

:G),

(672

:T),

(116

9:G

)(1

530:

G),

(156

1:A

),(1

574:

A),

(163

7:A

),(1

704:

A)

(199

1:I)

,(2

113:

C),

(246

7:T

),(2

472:

C),

(247

3:A

),(2

495:

A),

(255

7:A

)(2

663:

A),

(273

9:A

),(2

892:

A),

(311

1:T

),(3

116:

A)

Let

ter

desi

gnat

ion

sfo

rm

orph

ospe

cies

foll

owTa

ble

1.D

iagn

osti

cfe

atu

res

repr

esen

tu

niq

ue

com

bin

atio

ns

ofm

orph

olog

ical

char

acte

rsan

du

niq

ue

nu

cleo

tide

base

-pai

rch

ange

s(i

.e.

un

reve

rsed

auta

pom

orph

ies)

occu

rrin

gin

the

diag

nos

edta

xon

and

inn

oot

her

astr

oble

pid

orou

tgro

up

taxo

nex

amin

edin

this

stu

dy.

Seq

uen

cepo

siti

on:s

tate

spec

ifyi

ng

mol

ecu

lar

char

acte

rsgi

ven

inpa

ren

thes

es.

16S

,16S

rRN

A;A

,ade

nin

e;C

,cyt

osin

e;C

OI,

cyto

chro

me

cox

idas

esu

bun

itI;

Cyt

b,cy

toch

rom

eb;

G,g

uan

ine;

pmx,

prem

axil

lary

;Rag

-1,r

ecom

bin

atio

nac

tiva

tin

gge

ne

1;so

c,su

prao

ccip

ital

;T,

thym

ine.



molecular synapomorphies for species F is consistentwith the finding of paraphyly. Species G is a distinc-tive, but rare (undescribed) species known only fromtwo proximate collection sites separated by 2 km dis-tance in tributaries of the Río Consebidayoc of theupper Urubamba River drainage. It is diagnosedamongst morphospecies by the presence of distinctiveasymmetrically bifid teeth and absence of an adiposespine. These features are absent in representatives ofspecies F, which in turn is distinguished by a combi-nation of morphological characters (Table 2), none ofwhich alone represent apomorphies or featuresunique to morphospecies F. Our a priori hypothesisof species F distinction is not corroborated by thepresence of autapomorphic molecular characters.Although our samples of species F and G do notrepresent strictly sympatric populations, the twospecies nevertheless co-occur in a relatively short(21.4 km) section of the same upper Urubamba tribu-tary and therefore sympatry of these two species islikely (as occurs for multiple astroblepid species else-where in their distribution range) and could be testedupon additional fieldwork.

The case involving morphospecies H is even moresurprising, given the nature of its definition on thebasis of distinctive and unique morphological features(i.e. narrow mandibular ramus and wide, deep poste-rior lip; Table 2) and characteristic distribution inhigh-elevation streams. Although monophyly of thepopulation samples of species H was well supportedin both ML and MP analyses of the sequence data, wefound no apomorphic molecular characters withwhich to diagnose this species. As suggested by therelatively long branch length associated with thespecies H assemblage, this implies the presenceof numerous homoplastic (non-unique, reversed)changes in the molecular sequences in the lineageleading to the node inclusive of all species H samples(Fig. 3). Both species D and species H are occupantsof extreme headwater, high elevation habitats.Species H is known to occur at elevations from 2530to 3900 m within the Apurimac drainage, whereasspecies D has a much broader distribution range,known from 1500 to 4200 m elevation and occurringin both the Apurimac and Urubamba drainages. Theapparent allopatry of these sister species between theApurimac and Urubamba drainages, combined withthe presence of unique morphological characters inboth species, suggests that the absence of corroborat-ing molecular apomorphies in species H may be theresult of rapid divergence from a common ancestorshared with species D and insufficient time for fixa-tion of genetic differences between incipient species.Alternatively, this finding may represent little morethan our failure to capture the genomic divergencebetween species in the particular gene fragments

targeted by our analyses. These hypotheses, as wellas the proposition of separate status for species H,must be subjected to further analysis using additionalsources of data.

Although our phylogenetic analysis was restrictedto a small portion of the species diversity of the group(nine of approximately 70 species), a number of inter-esting phylogeographical patterns were discovered.First, those species sharing sympatric distributionswithin a particular drainage system were not alwaysrecovered as sister taxa. Species A, B, and C co-occurin multiple locations within the Madre de Dios riversystem and all three were collected at a single site inone particular tributary, the Araza River. In the phy-logenetic analyses, all three species were each recov-ered as more closely related to species assemblageswith representatives inhabiting river systems exter-nal to the Madre de Dios (i.e. the Marañon andApurimac/Urubamba, respectively) than to otherMadre de Dios species. Second, we recovered threeindependent instances of sister-group relationshipinvolving species distributed in both the Apurimacand Urubamba rivers (species D+H, F, E). We discusseach of these patterns in turn.

In the ML analysis (Fig. 3), species A was recoveredas sister to a representative of a species from theMarañon River, collected from a locality well outsidethe study region and separated by some considerablegeographical distance to the north-west. That speciespair is most closely related to species I, althoughrecovered without strong support. This result sug-gests broader clade membership of at least a portionof the southern astroblepid fauna. In the MP analysis,the inter-relationships amongst these three specieswas not resolved. Both species A and I were eachrepresented in our phylogenetic analysis by a singlesample. Species A is known from four localities and atotal of 30 preserved specimens, whereas species I isknown from four localities and a total of four speci-mens. Although we would obviously prefer to judgespecies validity on the basis of more complete sam-pling of these morphospecies, we note nevertheless arelatively large number of unique and unreversedmolecular sequence changes as additional support forthe recognition of these two species (Table 2). SpeciesA differs from all congeners in the study region in thepresence of highly distinctive chisel-shaped sym-metrically bifid jaw teeth, whereas species I differsfrom congeners in the presence of a highly distinctiveadipose fin, spine configuration, and bicoloured pig-mentation (Fig. 1P; dusky above lateral line, palebelow). Samples of both species are associated withrelatively long branch lengths in the ML tree (Fig. 3).

Species C (Madre de Dios) was recovered (althoughwith low support) as the sister group to a well-supported clade comprised of species E+F+G



(Apurimac+Urubamba rivers), with species B (Madrede Dios) recovered as the sister group to that assem-blage. Species D (Urubamba) and H (Apurimac) wererecovered as sister species and that clade wasstrongly supported as the sister group to all otheringroup species. Separate reciprocal geographicalclades (Apurimac, Urubamba) were recovered for thepopulation samples of species E and F+G, althoughwithout strong support in all analyses. Species Einhabits low to middle elevations, occurring from689 m in the Urubamba drainage to 2297 m in theApurimac. Urubamba representatives of species Einhabited small streams, whereas Apurimac repre-sentatives were found along the margins of largerrivers. Species F also occurred largely below 2300 mto as low as 560 m in the Urubamba, although a fewrecords in the Apurimac exceeded 2500 m elevation(e.g. as high as 2643 m in Sotccomayo/Pincus River).Intraspecific coloration pattern in species F variedwidely in the Urubamba, from uniform grey or brown(Fig. 1M) to boldly mottled or marbled with reddish-orange undertones (Fig. 1N). High levels of variationare perhaps most exemplified by the presence of thefull range of coloration patterns exhibited by speci-mens collected together at a single location (e.g.ANSP 180594, 180601; images showing additionalexamples of intraspecific variation in coloration arearchived at http://silurus.acnatsci.org/ACSI/field/Peru2004/fish/Astroblepidae/index_22-36.html).

Our results provide independent character evidencethat support the hypothesis of morphospecies in sevenof nine cases represented in the study area. Theseresults, evaluated within the context of the distribu-tion of the species, further indicate that astroblepidspecies are typically restricted in geographical distri-bution and endemic to single or adjacent riversystems of the Andes Mountains. As also observed forthe astroblepid fauna of the northern and centralportions of the Andean Cordilleras (e.g. Astroblepusorientalis, Astroblepus phelpsi, Astroblepus frenatus;Schaefer, 2003), species distributions generally do notcross the major headwater divides amongst drainagebasins (e.g. those separating the Ucayali and Madrede Dios watersheds), many of which involve eleva-tions above the altitudinal limits of the Andean fishfauna. Likewise, astroblepid species are limited at theopposite, lower extreme of their altitudinal range byecological conditions and physiological limits to life inwarm water (Schaefer, 2011). Of the six speciesendemic to the Ucayali watershed, only three species(D, E, and F) have relatively broader distributionsthat include both the Apurimac and Urubamba drain-ages within the more inclusive Ucayali system. Con-strained distributions at both extremes of theelevation range combine to limit astroblepid speciesto drainage islands within the Andean cordilleras,

thereby promoting isolation and divergence on rela-tively small spatial scales. The temporal scales ofastroblepid divergence and speciation have yet to bedirectly examined in detail.

These observations combine to suggest that thecurrent distribution of astroblepid species in thesouthern region may have arisen via a complexhistory involving both divergence between and dis-persal among drainage basins that is probablyrepeated numerous times throughout the Andean dis-tribution of the group. Upon inclusion in future analy-ses of additional representatives of species from othergeographical regions, we would expect to recoveradditional clades and expanded sets of relationshipsamongst groups of species beyond those recovered inthis limited analysis. The sorting of populationsamples by drainage within morphospecies (E, F)indicates that these particular species should bere-evaluated for the presence of undetected morpho-logical differences that are potentially congruent withthe observed geographical pattern of divergencewithin species.

ACKNOWLEDGEMENTS

We are grateful to Mariangeles Arce, Luis Fernández,Hernán Ortega, Lúcia Rapp Py-Daniel, NormaSalcedo, Leandro Sousa, and the students and staff ofthe Museu de Universidad Nacional Mayor de SanMarcos, Lima, for their assistance and participationin fieldwork activities in Peru. Robert Driver andKevin Geneva of the Laboratory for Molecular Sys-tematics and Ecology at the Academy of NaturalSciences provided laboratory assistance, as didMatthew Davis at LSU. We thank Jairo Arroyave,Robert Schelly and John Sparks for technical assis-tance, valuable comments, and discussion. Financialsupport was provided by the All Catfishes SpeciesInventory (NSF DEB 0315963), by an LMSE@ANSPsmall grant to M. Sabaj Pérez, and by NSF awardsDEB 0916695 to P. Chakrabarty and DEB 0314849 toS. Schaefer.

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SUPPORTING INFORMATION

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Appendix S1. Material examined.

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