The Fitzcarrald Arch: A Vicariant Event for Chaetostoma (Siluriformes:

Loricariidae) Speciation?

N. J. Salcedo1,2, D. Rodriguez1,3, R. E. Strauss1, and R. J. Baker1

The genus Chaetostoma is a species-rich group of armored catfishes that inhabits rivers and streams in montane forestsof South America. Most species of Chaetostoma have been described based on only one or a few specimens from a singleriver system. Our aim was to determine if the lifting of the Fitzcarrald Arch, 4 Ma, triggered speciation between thedisjunct populations of Chaetostoma lineopunctatum in two isolated river drainages: the Rıo Ucayali and the Rıo Madre deDios. We addressed this question by quantifying morphological and molecular divergence among samples of C.lineopunctatum from four river systems, three of which drain into the Rıo Ucayali drainage, including the type localityfor the species, and one river system which drains into the Rıo Madre de Dios drainage. Morphological divergence, basedon measurements, was statistically significant in the MANOVAs for the samples grouped by locality (P , 0.025) and byriver system (P , 0.025). However, neighbor-joining trees of specimens grouped by locality (based on Mahalanobisdistances) did not show group patterns according to river system. Morphologically, the specimens can be discriminatedby river system with up to 88.7% confidence based on size-free discriminant analysis. Furthermore, the divergencevalues among haplotypes from the Rıo Ucayali and the Rıo Madre de Dios drainages, based on partial cytochrome bsequences (801 bp), ranged from 1.1% to 1.5%, which overlaps the 1.2% divergence between haplotypes from the RıoUcayali drainage. Based on the available morphological and molecular data, we conclude that there are not significantdifferences among populations of C. lineopunctatum from the Rıo Ucayali and the Rıo Madre de Dios drainages, and thusthe Fitzcarrald Arch uplift did not cause speciation in this taxon. Alternatively, we suggest that isochronous tectonicevents prior to the uplift of the Fitzcarrald Arch could have triggered genetic isolation of populations of C.lineopunctatum.

AMONG the armored catfishes that inhabit thefreshwaters of South America, one group standsout for its distribution and habits: the genus

Chaetostoma. Chaetostoma includes 45 valid species (Fisch-Muller, 2003; Ferraris, 2007), of which 43 are distributedalong the Andean slopes from Panama to southern Peru andtwo are known to inhabit drainages of the Guyana Shield. Inaddition, 42 species have been described based on one orfew specimens from a single river system, which could beinterpreted as a high number of species with restricteddistributions. Furthermore, many lots of Chaetostoma de-posited in museum collections are identified as undeter-mined species of Chaetostoma (Chang, 1998; Ortega et al.,2001; Willink et al., 2005), which reflects the dearth ofknowledge about the taxonomy of this genus. The highnumber of rare species could also be an artifact attributed tolimited access to habitats.

In Peru, considerable efforts have promoted the collectionof fish fauna in remote areas during the last ten years(Ortega and Hidalgo, 2008). These efforts include RapidAssessment Inventory programs that target candidate areasfor conservation (Chernoff et al., 2000; Hidalgo and Quispe,2004; Hidalgo and Silva, 2006), and the baseline studiesrequired by the Peruvian Government for exploration,exploitation, and/or extraction of natural resources (Chang,1998; Ortega et al., 2001). In the context of baseline studies,southeastern Peru has received special attention because ofthe presence of important oil and natural gas reservoirs inSouth America (Espurt et al., 2007). Based on the currenttaxonomic knowledge of the Peruvian fish fauna, research-ers have suggested the possibility of finding undiscoveredand undescribed species of freshwater fishes in remote andisolated highland areas (Ortega and Hidalgo, 2008), such as

the slopes of the Fitzcarrald Arch (Maxime and Albert, 2009;Espurt et al., 2010), an elevational feature (400 to 600 m asl)that separates the foredeeps of the northern and southernAmazonian foreland basins (Regard et al., 2009).

The geological and physiographical history of southeast-ern Peru is particularly complex, and as such, few events canbe pinpointed in the history of its river basins. One eventthat has been documented is the capture of the headwatersof the Rıo Madre de Dios. The headwaters of the Rıo Madrede Dios changed their south to north direction to a north tosouth direction when the Nazca Ridge produced thelithosphere uplift with the subsequent dissection of theEastern Cordillera (central Andes) at around 4 Ma (Espurt etal., 2007; Regard et al., 2009). Consequently the water flowand fauna of the upper Rıo Madre de Dios became part of theMadeira River drainage (Mora et al., 2010; Roddaz et al.,2010; Fig. 1). Thus, considering the uplifting of the regionlifted by the Nazca Ridge as a vicariant event (known as theFitzcarrald Arch, but see Wesselingh and Salo [2006:452] fordisambiguation on the use of this term), this river captureproduced the isolation of the freshwater populations in theRıo Ucayali and Rıo Madre de Dios drainages as early as 4 Ma,which could be correlated with cladogenesis and ultimatelyto speciation events (Espurt et al., 2010).

The species C. lineopunctatum was described based on twospecimens from the Rıo Azupizu, a tributary of the RıoPachitea, Rıo Ucayali drainage in central Peru (Eigenmannand Allen, 1942). This species is distinguished from all otherspecies of Chaetostoma based on the presence of small blackdots on the body and head, and two series of black dots onthe membranes between its dorsal-fin rays. Specimens fromseveral river systems that drain into the isolated Rıo Ucayaliand Rıo Madre de Dios drainages possess the diagnostic

1 Department of Biological Sciences, Texas Tech University, Flint and Main, Lubbock, Texas 79409-3131.2 Present address: Grice Marine Laboratory, College of Charleston, 205 Fort Johnson, Charleston, South Carolina 29412; E-mail:

nj.salcedo@gmail.com. Send reprint requests to this address.3 Present address: Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, New York 14853.Submitted: 9 April 2010. Accepted: 2 September 2011. Associate Editor: R. E. Reis.F 2011 by the American Society of Ichthyologists and Herpetologists DOI: 10.1643/CI-10-062

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characters of C. lineopunctatum, but there may be species-level differences between the populations that inhabitdifferent river systems. Thus, the aim of this study is to testif the uplift of the Fitzcarrald Arch triggered speciationbetween the Rıo Madre de Dios and the Rıo Ucayalipopulations of C. lineopunctatum. Our null hypothesis isthat there are no significant differences between popula-tions of C. lineopunctatum from the Rıo Madre de Dios andthe Rıo Ucayali drainages. This question is addressed byquantifying the morphological and molecular divergencesof populations of C. lineopunctatum from central andsoutheastern Peru.

MATERIALS AND METHODS

Specimens.—Specimens were collected from two drainages:Rıo Ucayali and Rıo Madre de Dios. The Rıo Tambo and RıoUrubamba systems drain into the Rıo Ucayali drainage, andthe Rıo Inambari system drains into the Rıo Madre de Diosdrainage. For the analyses, specimens were grouped bylocality of collection within river systems: two localities inthe Rıo Tambo system (Rıo Perene, Rıo Pichanaki), threelocalities in the Rıo Urubamba system (Rıo Coribeni, RıoIgoritoshiari, Rıo Manogali), and three localities in the RıoInambari system (Quebrada Huajidumbre, Rıo Inambari, RıoNusiniscato). Museum specimens from two localities of the

Rıo Pachitea system (Rıo Pozuzo, Puerto Bermudez) werestudied and included in the morphometric analysis. Allspecimens were fixed in 10% formalin and transferred to70% ethanol after 48 hours. Tissue samples (muscle and/orgill rakers) were obtained from fresh specimens in the fieldand preserved in 95% ethanol or lysis buffer (Longmire etal., 1997). All extracted DNA samples are deposited in theNatural Science Research Laboratory of Texas Tech Univer-sity (NSRL). Collection data for all the specimens arepresented in the Material Examined section. The name-bearing type of the species C. lineopunctatum, in this case theholotype, was included in the present study to comply withthe objectivity provided by typification (ICZN, Article 61.Principle of Typification) towards the stability of scientificnames (ICZN, 1999).

Morphometric analysis.—Linear measurements were ob-tained from each specimen based on 29 landmarks(Fig. 2) using digital calipers on the left side of the body,unless the structure in question was damaged, in whichcase the measurement was made on the right side. A set of35 measurements was obtained as described in Appendix 1.Trunk measurements associated with paired-fins insertion(pectoral–pelvic, pelvic–anus, and pelvic–anal lengths)were estimated as an average of right and left sides. All

Fig. 1. Map of Peru showing localities from where specimens ofChaetostoma lineopunctatum included in the present study werecollected. Open symbols, specimens with no associated tissue samples;solid symbols, specimens with associated tissue samples. Type locality(star); Rıo Pachitea system (squares); Rıo Tambo system (diamonds);Rıo Urubamba system (circles); and Rıo Inambari system (triangles).

Fig. 2. Landmarks for morphometric analysis. (A) Lateral view: 1,anterior tip of the mesethmoid; 2, posterior margin of bony nostril; 3,anterior margin of bony orbit; 4, posterior margin of bony orbit; 5,posterior tip of supraoccipital; 6, anterior margin of first dorsal-fin ray atbase; 7, tip of first dorsal-fin ray; 8, posterior margin of last dorsal- fin rayat dorsal-fin base; 9, tip of last dorsal-fin ray; 10, posterior margin ofpreadipose plate; 11, tip of adipose spine; 12, anterior margin of lastdorsal procurrent ray; 13, posterior margin of the last median lateralplate; 14, posterior margin of last anal-fin ray at base. (B) Dorsal view: 15,medial margin of bony nostril; 16, medial margin of bony orbit; 17, lateralmargin of exposed cleithrum. (C) Ventral view: 18, medial margin ofpremaxilla; 19, lateral margin of premaxilla; 20, medial margin of dentary;21, lateral margin of dentary; 22, medial edge of branchial opening; 23,anterior margin of pectoral spine at base; 24, bony tip of pectoral spine;25, anterior margin of first pelvic-fin ray; 26, tip of first pelvic-fin ray; 27,base of urogenital papilla; 28, anterior margin of first anal-fin ray at base;29, anterior margin of last ventral procurrent ray.

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mensural data were transformed to natural logarithms foranalysis. Missing data due to damaged specimens wereestimated by using the maximum likelihood expectation–maximization algorithm (Dempster at al., 1977; Little andRubin, 1987). The specimens studied do not exhibit thesecondary sexual characteristics known for this genus(Rapp Py-Daniel, 1991), and all of them are treated as ofundetermined sex.

To determine the best subset of measurements thatexplained the differences among samples, all measurementswere included in a stepwise discriminant analysis, whichintroduces variables at each step so as to maximize theLawley-Hotelling trace (Rao’s V or Hotelling’s generalizedT2). From this analysis, variables were ranked by theircontribution to discrimination, and a subset of the mostdiscriminatory ones was included in the multivariateanalysis of variance (MANOVA) for specimen groups.

To determine the degree of statistical significance of thedifferences among specimens grouped by locality and bybasin, two separate MANOVAs were performed. The numberof variables included in each MANOVA was determined bythe maximum number of variables that did not return asingular matrix in the MANOVA analysis. The same subsetof variables included in the MANOVAs was used in theanalyses described below.

Mahalanobis distances based on the pooled covariancematrix among all specimen groups (Mahalanobis, 1948)were estimated for all samples (excluding small ones). Basedon these distance values, clusters were estimated by findingan additive tree network using the Saitou and Nei (1987)neighbor-joining method, simplified as in Studier andKepler (1988). This method uses the Kuhner and Felsenstein(1994) calculations to adjust negative branch lengths. As thenetwork has no specified outgroup, Farris’ (1972:658)minimum rate-heterogeneity criterion is used to estimatethe root, which returns an ancestor function and vector ofcorresponding branch length. Covariance matrices of allthese samples were bootstrapped and the values are reportedon the corresponding phenogram.

To determine linear functions that could be used foridentifying groups, discriminant functions for specimensgrouped by basin were calculated based on size-invariant(‘‘size-free’’) measurements, where the log-transformedvalues of each character are regressed separately on the firstpooled within-group principal component (Reis et al., 1990;Oliveira, 1998).

All morphometric analyses were performed using programswritten in Matlab (Mathworks, http://www.mathworks.com/products/matlab/), mainly on the library functions developedby Richard Strauss (available at http://www.faculty.biol.ttu.edu/Strauss/Matlab/Matlab.html).

Molecular analysis.—Genomic DNA from tissue samples wasextracted following Longmire et al. (1997). Two primerswere used to amplify the complete cyt b gene: GluDG.L59–TGA CCT GAA RAA CCA YCG TTG–39 (modified fromPalumbi, 1996) and H16460 59–CGA YTC TCG GAT TACAAG ACC G–39 (Perdices et al., 2002), with complementarysequences on the glutamine tRNA and threonine tRNAflanking regions, respectively. One internal primer was usedwith GluDG.L to amplify an approximately 900-bp fragmentof the cyt b gene on all specimens from the Rıo Urubambabasin: CB3.H 59–GGC AAA TAG GAA RTA TCA TTC–39

(Palumbi, 1996). PCR products were produced in 25 ml

reactions, using 5 ml 53 Colorless Go TaqH Flexi Buffer, 3 ml25 mM MgCl2, 0.25 ml GoTaqH FlexiDNA polymerase(Promega), 2.5 ml 2.5 mM dNTP, 1.5 ml 10 mg/ml BSA, 1 mlof each 10 mM primer, 1–5 ml template DNA, and water. ThePCR conditions follow Perdices et al. (2002). DNA sequenceof the first 810 bp of cyt b gene was obtained from eachpurified PCR product through double-stranded sequencingusing GluDG.L and CB3.H as flanking primers and CB450.L59–TAC ATC GGA RAC AAC CTA G–39 and CB2.H 59–CCCTCA GAA TGA TAT TTG TCC TCA–39 (Palumbi, 1996) asinternal primers.

The complete curated cyt b gene sequences for Cranoglanisbouderius (Peng et al., 2006) and Ictalurus punctatus (Wald-bieser et al., 2003) were obtained from GenBank and usedfor alignment, performed using AlignX software, as nocurated sequences of closer relatives were available at thetime this analysis was ongoing. Sequences of Chaetostomaloborhynchos were included in the study as a closely relatedoutgroup for the phylogenetic analysis. All sequences werealigned and trimmed to the shortest available length. Thesequences obtained were translated into predicted amino-acid sequences using MacClade 4.06 (Maddison and Maddi-son, 2003) to evaluate for the presence of stop codons.

A haplotype network was constructed using TCS 1.1(Clement et al., 2000). Phylogenetic relationships wereestimated using both maximum parsimony and maximumlikelihood criteria using PAUP* v. 4.0.b10 (Swofford, 2001).For the maximum likelihood criterion, the best-fittingmodel of evolution for the data was estimated usingjModelTest (Posada, 2008). Nodal support for the maximumparsimony and maximum likelihood analyses were estimat-ed using nonparametric bootstrapping (1000 pseudo-repli-cates). The sequences were also tested for clock-like behaviorof substitution rates among lineages by determining alikelihood score for the best topology with and withoutconstraint of a molecular clock; the scores obtained wereused in a log-likelihood test (Drake et al., 2007).

RESULTS

Morphometric analysis.—The stepwise discriminant analysisindicated an increase of discrimination as variables wereadded for the 35 measurements, with a significant increasein discrimination per variable until the addition of the fifthvariable, and with smaller increases of discrimination pervariable as more variables are added. This corresponds to atrend of small decrease of the values for discrimination byvariable (discrimination divided by the number of variablesincluded at each step) after the addition of the 15th variable.Thus, the 15 most discriminatory measurements for allspecimens studied (from most discriminatory to leastdiscriminatory: internostril width, pectoral spine length,dentary length, interpelvic width, snout length, caudalpeduncle length, interorbit width, pre-anal length, adiposespine length, pelvic–anus length, first dorsal-fin ray length,adipose–caudal length, bony orbit length, cleithral width,base of dorsal fin length) were included in the MANOVAs,which indicate levels of statistical significance amongspecimens grouped by locality (P , 0.025) and by basin(P , 0.025; Table 1).

Mahalanobis distances of the pooled covariance matrix ofsize-free measurements among specimen groups from eightlocalities (Table 2), represented as a neighbor-joining tree(Fig. 3), show the specimens grouped as follows: RıoCoribeni (Rıo Urubamba) and Rıo Perene (Rıo Tambo) are

Salcedo et al.—Chaetostoma and the Fitzcarrald Arch 505

paired, Rıo Igoritoshiari (Rıo Inambari), Puerto Bermudez(Rıo Pachitea), Rıo Pichanaki (Rıo Tambo), Rıo Pozuzo (RıoPachitea), Rıo Nusiniscato (Rıo Inambari), and QuebradaHuajidumbre (Rıo Inambari) are successively grouped to thispair. Bootstrap values are significant (.50%) only for thepair Quebrada Huajidumbre–Rıo Nusiniscato (Rıo Inambari),although this pair does not appear as a group in thetopology found. The specimens grouped by localities didnot pair according to river system.

From the size-free discriminant analyses the first twodiscriminant functions account for 65.5% and 88.7%

discrimination among groups by locality and by system,respectively. Plots of scores for the first two size-freediscriminant functions (SF-DF1 vs. SF-DF2) for specimensgrouped by localities and by system also show scatters thatoverlap among groups but are significantly heterogeneous(Fig. 4).

Molecular analysis.—The complete cyt b gene sequence for C.lineopunctatum is 1134 bp long, from a presumed start codon(ATG) to a stop codon. The first 801 bp, which correspond to267 amino acids, were included in the analysis. Twohaplotypes were found among individuals from the RıoUcayali drainage and four haplotypes among individualsfrom the Rıo Madre de Dios drainage (Table 3). A haplotypenetwork showed three major haplogroups among popula-tions from the Rıo Madre de Dios and the Rıo Ucayalidrainages (Fig. 5). The divergence among haplotypes fromthe Rıo Madre de Dios drainage ranged from 0.1% to 0.4%.The divergence between haplotypes from the Rıo Tamboand the Rıo Urubamba systems, both in the Rıo Ucayalidrainage, was 1.2%. The divergence between haplotypesfrom the Rıo Ucayali and Rıo Madre de Dios drainagesranged between 1.1% and 1.5% (Table 4).

The aligned sequences were arranged in a data matrix thatincluded 801 characters (bp) for 26 specimens. The TIM2+Imodel was selected as the most appropriate model of

evolution for these data for the maximum likelihoodanalysis. Parsimony and maximum likelihood criteria bothretrieved the same topology (Fig. 6): all the Rıo Inambarihaplotypes grouped in one clade, closely related to the RıoTambo haplotype. The Rıo Urubamba haplotype is basal toall other haplotypes. There were not significant differencesin rates of substitution among lineages (x2 5 25.22; df 5 25;P 5 0.395); thus, the hypothesis of a clocklike behaviorcannot be rejected.

DISCUSSION

The significant MANOVAs by locality and by river systemcould indicate incipient morphological differentiation be-tween populations from the Rıo Madre de Dios (RıoInambari localities) and the Rıo Ucayali drainages (RıoPachitea, Rıo Tambo, and Rıo Urubamba localities), but thiscould also be the result of ecophenotypy. Moreover, if weidentified specimens using two discriminant functions, only89 out of 100 would be correctly assigned to a river system(Rıo Pachitea, Rıo Tambo, Rıo Urubamba, or Rıo Inambari),possibly because the morphological differences amongsample groups are so small that the discrimination amongthem is dampened out.

Although the haplotypes for the populations of C.lineopunctatum studied are unique to the Rıo Inambari, RıoTambo, and Rıo Urubamba systems, the genetic divergencebetween haplotypes among these populations is small (1.2%

between Rıo Urubamba and Rıo Tambo; 1.1%, 1.4%, and1.5% between Rıo Inambari and Rıo Urubamba; and 1.1%,1.4%, and 1.5% between Rıo Inambari and Rıo Tambo)compared to the 8.6% divergence between the sympatric C.lineopunctatum and C. loborhynchos in the Rıo Perene(Salcedo, 2007). Furthermore, the absence of morphologicalattributes that alone or in combination can provide aunique set of diagnostic characters to identify each of thesepopulations as distinct species leads us to recognize all the

Table 2. Mahalanobis Distances among Chaetostoma lineopunctatum Specimen Groups from Eight Localities Based on Size-freeMorphometric Data.

Drainages Rıo Ucayali Rıo Madre de Dios

Systems Rıo Pachitea Rıo Tambo Rıo Urubamba Rıo Inambari

Localities 1 2 3 4 5 6 7 8

1. Puerto Bermudez – 35.69 30.31 28.05 52.98 34.06 48.15 33.182. Rıo Pozuzo – 37.23 13.59 39.04 29.69 20.47 17.013. Rıo Perene – 15.96 26.33 34.80 57.26 57.154. Rıo Pichanaki – 33.69 18.56 21.98 21.655. Rıo Coribeni – 22.71 44.35 52.996. Rıo Igoritoshiari – 22.47 15.577. Quebrada Huajidumbre – 11.568. Rıo Nusiniscato –

Table 1. Results from Multivariate Analyses of Variance (MANOVA) of 15 Most Discriminatory Log Morphometric Variables, Partitioned by Localityand by System.

Wilk’s ldf among

groupsdf within

group df total F df 1 df 2 P-value

By locality 0.0022 9 116 125 7.1431 135 808 ,0.001By system 0.0469 3 122 125 12.8437 45 321 ,0.001

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studied populations as members of a single species. Thus, C.lineopunctatum is the species with the widest documenteddistribution among species of Chaetostoma.

Based on our results we cannot reject our null hypothesis,that there are no significant differences between popula-tions of C. lineopunctatum from the Rıo Madre de Dios andthe Rıo Ucayali drainages, and we conclude that theFitzcarrald Arch uplift and the subsequent river capture ofthe headwaters of the Rıo Madre de Dios 4 Ma did not triggervicariant speciation in the ancestral species. Additionally,based on the haplotype network with similar distancesamong haplogroups, and the phylogenetic pattern, which isincongruent with the lifting of the Fitzcarrald Arch as avicariant event, we suggest that these populations couldhave separated from each other at around the same time andbefore the change of direction of the Rıo Madre de Dios. Thishypothesis is based on the observations of the first authorthat suggest the dispersal of individuals of species ofChaetostoma might be limited by the physical barrier formedby the deposition of fine sediments associated with lowenergy or deltaic environments downstream.

The uplift of the Andes is an ongoing process that can betraced back to the Late Cretaceous (Lundberg et al., 1998).However, there were pulses of violent lifting during theMiocene that are related to the erosion of the eastern slopeof the Andes and the deposition of sediments, which havebeen recognized in studies of the stratigraphy of Amazonia(Campbell et al., 2006). If the populations of C. line-opunctatum in the Rıo Inambari, Rıo Tambo, and RıoUrubamba systems were separated by isochronous eventsearlier than 4 Ma, then it is possible that a violent lifting ofthe Eastern Cordillera could be associated with thisseparation. Then, the Quechua II orogenic pulse, whichended the Ucayali peneplanation in the Late Miocene ataround 9 Ma, could be responsible for the elevation of theEastern Cordillera (Benavides-Caceres, 1999) that isolatedthe populations of C. lineopunctatum in these river systemsby the depositional Rıo Ucayali.

Although this work includes abundant material of C.lineopunctatum, the knowledge on the distribution of thisspecies is still incomplete, as this study is based only onavailable material obtained from the most accessible areas.The study of all specimens of C. lineopunctatum in museumcollections is lacking, and it is necessary for further

discussion of the distribution of the species. Ecological andphysical limitations appear to preclude the presence ofspecies of Astroblepus in lower courses of rivers (Schaefer andArroyave, 2010). However, there are no studies about thedispersion patterns of C. lineopunctatum and their compar-ison to those of species of Astroblepus. Considering theAndes mountain range as a system of lifting islands with fishriding on them (Schaefer, 2011), the phylogenetic patternsof divergence should be engraved in the signal thatmorphological or molecular analyses retrieve for unrelatedtaxa that inhabit these environments. In this respect,molecular data for Astroblepus (Schaefer et al., 2011) andfor Chaetostoma (present study) in southeastern Peru arecompatible, showing a basal clade of specimens from the Rıo

Fig. 4. Plots of scores for the first two discriminant functions for size-free data (SF-DF1 vs. SF-DF2) for specimens of Chaetostoma line-opunctatum grouped by (A) locality and (B) system.

Fig. 3. Neighbor-joining tree based on Mahalanobis distance values ofsize-free data for specimens of Chaetostoma lineopunctatum fromeight localities in four river systems.

Salcedo et al.—Chaetostoma and the Fitzcarrald Arch 507

Madre de Dios system and two sister clades of specimensfrom the Rıo Ucayali system, which points to a complexsequence of events that may have caused the separation ofthese fish populations in southeastern Peru. We expect thisphylogenetic pattern to be reflected in other taxa distributedin the fast flowing waters of the high Andes.

MATERIAL EXAMINED

Institutional abbreviations are as listed by Sabaj Perez(2010). Additionally, NSRL stands for Natural ScienceResearch Laboratory at the Museum of Texas Tech Univer-sity, Texas. The specimens included in this study are listedby river system, including lot catalog number, number ofspecimens, size range, locality, collector, and collectiondate. If tissue samples were available from voucher speci-mens in the lot, the tissue sample number (P and TKcorrespond to ANSP and NSRL tissue collections, respective-ly) and GenBank accession number in parenthesis follow.

Chaetostoma lineopunctatum: Peru, Departamento de Pasco,Provincia de Oxapampa, Rıo Pachitea River system: CAS64650 (ex IU 15373), holotype, 110.77 mm SL, Rıo Azupizu,W. R. Allen, July 1920; MUSM 1801, 1, 47.01 mm SL, VillaAmerica, Rıo Maız, P. Hocking, September 1983; MUSM17226, 1, 54.37 mm SL, Santa Rosa (500 m), Rıo Palcazu, P.Hocking, 30 September 1988; MUSM 19301, 1, 68.31 mmSL, Pto. Bermudez, Qda. Km. 8, 10u20912.60S, 74u59916.50W,H. Ortega et al., 9 July 2001; MUSM 20568, 1, 45.73 mm SL,

Pto. Bermudez, Qda. Atas, 10u21952.30S, 74u58946.20W,Earthwatch Team III, 9 August 2002; MUSM 20581, 1,53.65 mm SL, Pto. Bermudez, Qda. Soldado, 10u22925.70S,74u59900.60W, Earthwatch Team III, 9 August 2002; MUSM26563, 2, 66.0–70.51 mm SL, Yanahuanca, Rıo Sta. Cruz, E.Castro et al., 20 October 2005; MUSM 26572, 7, 25.93–48.71 mm SL, Pozuzo, Sta. Rosa, Rıo Pozuzo, E. Castro et al.,21 October 2005; MUSM 26579, 1, 34.6 mm SL, Pozuzo, RıoPozuzo, E. Castro et al., 21 October 2005; MUSM 26582, 2,33.42–35.4 mm SL, Pozuzo, Qda. Ceso, 10u01949.70S,75u31950.90W, E. Castro et al., 21 October 2005. Peru,Departamento de Huanuco, Provincia de Puerto Inca, RıoPachitea basin: MUSM 10621, 8, 30.81–59.08 mm SL,Pachitea, Yuyapichis, 2 km from mouth, 9u37953.90S,74u56954.50W, E. Holm et al., 29 July 1988; MUSM 12799,2, 26.64–44.05 mm SL, Pachitea, Yuyapichis, Qda. 1.8 km Eof Panguana, 09u369250S, 74u55.570W, E. Holm, 27 July1988; MUSM 12905, 1, 40.86 mm SL, Pachitea, Rıo Pachitea,mouth of Rıo Huambo, 9u399160S, 74u569220W, E. Holm, 28July 1988. Peru: Departamento de Junın, Provincia deSatipo, Rıo Tambo System: MUSM 20301, 15, 46.82–70.7 mm SL, Pichanaki, Rıo Pichanaki, 10u559460S,74u53900.70W, N. Salcedo and B. Sidlauskas, 8 August2003, TK70564 (GU569908), TK70566 (GU569909),TK70567 (GU569910); MUSM 20305, 7, 15.15–67.65 mmSL, Pichanaki, Rıo Perene, 10u53926.20S, 74u56950.30W, N.Salcedo and B. Sidlauskas, 8 August 2003, TK70568(GU569911), TK70571 (GU569912). Peru, Departamentode Cusco, Provincia de La Convencion, Rıo Urubambasystem: ANSP 180442, 6, 30.8–79.3 mm SL, Coribeni, RıoCoribeni, 12u37921.30S, 72u50908.60W, TCEP04-32, 20 July2004, TK94299 (GU569901), TK94300 (GU569902),TK94301 (GU569903); ANSP 180446, 1, 57.06 mm SL,Monte Carmelo, Rıo Manogali, 12u36916.00S, 73u089

13.10W, TCEP04-38, 21 July 2004, P4772 (GU569899); ANSP180447, 10, 22.82–67.79 mm SL, Rıo Igoritoshiari,12u26956.10S, 73u00958.90W, TCEP04-37, 21 July 2004,TK94305 (GU569904), TK94306 (GU569905), TK94307(GU569906), TK94308 (GU569907); AUM uncat., 10,17.37–52.78 mm SL, Rıo Igoritoshiari, 12u26956.10S,73u00958.90W, TCEP04-37, 21 July 2004; INPA uncat., 10,18.62–57.68 mm SL, Rıo Igoritoshiari, 12u26956.10S,73u00958.90W, TCEP04-37, 21 July 2004; MUSM uncat., 4,29.96–69.94 mm SL, Coribeni, Rıo Coribeni, 12u37921.30S,72u50908.60W, TCEP04-32, 20 July 2004; MUSM uncat.,30, 15–60.2 mm SL, Rıo Igoritoshiari, 12u26956.10S,73u00958.90W, TCEP04-37, 21 July 2004; MUSM uncat.,

Table 3. Variable Positions in the 801 bp Cytochrome b Gene Segment of Chaetostoma lineopunctatum from the Rıo Ucayali and Rıo Madre de DiosDrainages. Dots represent identical nucleotides to Haplotype A.

Haplotypes

No. individualsat eachdrainage

Position

424

124

196

231

282

291

324

345

405

423

435

465

469

489

690

738

789

Rıo Madrede Dios

RıoUcayali

A (7) A C A G T T C C T C G C C A T G T CB (1) . . . A . . . . . . . . . . . . . .C (1) . . . . C . . . . . . . . . . . . .D (1) . . . . . . . . C . . . . . . . . TE (9) . . G . . C . T C T . T T . C A C TF (5) G A G . . C T . C T A T . G . . . T

Fig. 5. Haplotype network of cytochrome b sequences from specimensof Chaetostoma lineopunctatum collected from three river systems incentral and southeastern Peru. Nodes sizes are proportional tohaplotype frequency.

508 Copeia 2011, No. 4

1, 38.01 mm SL, Monte Carmelo, Rıo Manogali, 12u36916.00S,73u08913.10W, TCEP04-38, 21 July 2004, P4773 (GU569900).Peru, Departamento de Cusco, Provincia de Quispicanchi, RıoInambari system: ANSP 180441, 8, 20.83–62.49 mm SL, Rıo

Nusiniscato, 13u11917.10S, 70u34905.10W, TCEP04-48, 26 July2004, TK94313 (GU569919), TK9414 (GU569920); ANSP180448, 7, 39.17–88.77 mm SL, Pte. Palcamayo, Qda.Huajidumbre, 13u12905.80S, 70u41903.40W, TCEP04-47, 26

Table 4. Genetic Divergence Values (Cytochrome b, 801 bp) among Haplotypes of Chaetostoma lineopunctatum from the Rıo Ucayali and RıoMadre de Dios Drainages. Above diagonal, percent differences; below diagonal, total character differences.

Drainages Haplotypes

Rıo Madre de Dios Rıo Ucayali

A B C D E F

Rıo Madre de Dios A – 0.125 0.125 0.250 1.373 1.373B 1 – 0.250 0.375 1.498 1.498C 1 2 – 0.375 1.498 1.498D 2 3 3 – 1.124 1.124

Rıo Ucayali E 11 12 12 9 – 1.248F 11 12 12 9 10 –

Fig. 6. Maximum likelihood tree based on cytochrome b sequences from specimens of Chaetostoma lineopunctatum from three river systems incentral and southeastern Peru. Lnl 5 21192.8221; Ti/tv ratio 5 17.1371; PA 5 0.2995; PC 5 0.2796; PG 5 0.1288; PT 5 0.2921; Pinv 5 0; equalrates for all sites. Numbers on branches denote bootstrap values. Bootstrap values are only shown when $50%.

Salcedo et al.—Chaetostoma and the Fitzcarrald Arch 509

July 2004, P4814 (GU569913), P4815 (GU569914), TK94309(GU569915), TK94310 (GU569916), TK94311 (GU569917),TK94312 (GU569918); AUM uncat., 4, 51.42–92.27 mmSL, Pte. Palcamayo, Qda. Huajidumbre, 13u12905.80S,70u41903.40W, TCEP04-47, 26 July 2004; INPA uncat., 4,42.62–89.67 mm SL, Pte. Palcamayo, Qda. Huajidumbre,13u12905.80S, 70u41903.40W, TCEP04-47, 26 July 2004; MUSMuncat., 15, 46.79–86.18 mm SL, Pte. Palcamayo, Qda.Huajidumbre, 13u12905.80S, 70u41903.40W, TCEP04-47, 26July 2004; MUSM uncat., 9, 16.65–62.94 mm SL, RıoNusiniscato, 13u11917.10S, 70u34905.10W, TCEP04-48, 26 July2004. Peru, Departamento de Madre de Dios, Provincia deTambopata, Rıo Inambari system: ANSP 180445, 1, 47.67 mmSL, Rıo Inambari, opposite margin from Rıo Quiniri’s mouth,13u05949.70S, 70u24911.50W, TCEP04-50, 27 July 2004,TK94315 (GU569921); MUSM uncat., 1, 44.21 mm SL, RıoInambari, opposite margin from Rıo Quiniri’s mouth,13u05949.70S, 70u24911.50W, TCEP04-50, 27 July 2004,TK94316 (GU569922).

ACKNOWLEDGMENTS

For field assistance in 2002, 2003, and 2004 we thank M. Arce,M. Arizaga, L. Chocano, E. Lopez, M. Lunberg, L. de Souza, H.Ortega, R. Quispe, L. Rapp-PyDaniel, B. Rengifo, M. Sabaj, B.Sidlauskas, and L. Sousa. For discussions we thank R. Bradley,L. Densmore, and J. Lundberg. For loans of specimens undertheir care and for facilitating information related to suchmaterial, thanks to E. Castro, D. Catania, E. Holm, M. Hoang,T. Iwamoto, J. Lundberg, V. Meza, H. Ortega, and M. Sabaj.For help with molecular data collection and DNA analysesthanks to S. Hoofer and P. Larsen. Thanks to W. Anderson, Jr.and A. Harold for valuable editorial suggestions on themanuscript. NJS visit to CAS was supported by the LakesideFoundation. Most tissue specimens were available thanks tothe All Catfish Species Inventory Project (NSF DEB-0315963,L. Page, P.I.). Collections in Peru were made under thepermits from the Peruvian Ministry of Fisheries R.D. No.122-2003-PRODUCE/DNEPP and R.D. No. 283-2004-PRODUCE/DNEPP.

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APPENDIX 1

DESCRIPTION OF POINT TO POINT MEASUREMENTS BASEDON 29 LANDMARKS FOR CHAETOSTOMA SPECIMENS.

1) Standard length (SL), from anterior tip of mesethmoidto posterior margin of last median lateral plate; 2) Headlength (HL), from anterior tip of mesethmoid to posterior tipof supraoccipital; 3) Head depth (HD), from posterior tip ofsupraoccipital to ventral surface of body in a straight angle;4) Body depth (BD), from anterior margin of first dorsal-finray at base to ventral surface of body in a straight angle; 5)Cleithral width (CW), from left to right lateral margins ofexposed cleithrum; 6) Interbranchial width (IBW), from leftto right medial edges of branchial openings; 7) Pre-dorsallength (PD), from anterior tip of mesethmoid to anteriormargin of first dorsal-fin ray at base; 8) Base of dorsal finlength (DB), from anterior margin of first dorsal-fin ray at

Salcedo et al.—Chaetostoma and the Fitzcarrald Arch 511

base to posterior margin of last dorsal-fin ray at dorsal-finbase; 9) Dorsal fin length (DL), from anterior margin of firstdorsal-fin ray at base to tip of last dorsal-fin ray; 10) Firstdorsal-fin ray length (FDR), from anterior margin of firstdorsal-fin ray at base to its tip; 11) Last dorsal-fin ray length(LDR), from posterior margin of last dorsal-fin ray at dorsal-fin base to its tip; 12) Dorsal–caudal length (DC), fromposterior margin of last dorsal-fin ray at dorsal-fin base toanterior margin of last dorsal procurrent ray; 13) Dorsal–adipose length (DAd), from posterior margin of last dorsal-fin ray at dorsal-fin base to posterior margin of pre-adiposeplate; 14) Adipose spine length (AdS), from posterior marginof pre-adipose plate to tip of adipose spine; 15) Adipose–caudal length (AdC), from posterior margin of pre-adiposeplate to anterior margin of last dorsal procurrent ray; 16)Pectoral–pelvic length (PPv), from anterior margin ofpectoral spine at base to anterior margin of first pelvic-finray at base; 17) Pectoral spine length (PS), from anteriormargin of pectoral spine at base to its bony tip; 18)Interpelvic width (IPv), from left to right anterior marginof first pelvic-fin ray at base; 19) First pelvic-fin ray length(PvFR), from anterior margin of first pelvic-fin ray at base toits tip; 20) Pelvic–anus length (PvUL), from anterior marginof first pelvic-fin ray at base to base of uro-genital papilla;21) Pre-anus length (PU), from anterior tip of mesethmoidto base of uro-genital papilla; 22) Anus–caudal length (UC),

from base of uro-genital papilla to anterior margin of lastventral procurrent ray; 23) Anus–anal length (UA), frombase of uro-genital papilla to anterior margin of first anal-finray at base; 24) Pelvic–anal length (PvAL), from anteriormargin of first pelvic-fin ray at base to anterior margin offirst anal-fin ray at base; 25) Pre-anal length (PA), fromanterior tip of mesethmoid to anterior margin of first anal-fin ray at base; 26) Anal–caudal length (AC), from anteriormargin of first anal-fin ray at base to anterior margin of lastventral procurrent ray; 27) Caudal peduncle length (CPL),from posterior margin of last ventral-fin ray at base toanterior margin of last ventral procurrent ray; 28) Caudalpeduncle depth (CPD), shallowest depth of the caudalpeduncle in a straight angle; 29) Snout length (SnL), fromanterior tip of mesethmoid to anterior margin of bony orbit;30) Internostril width (IN), shortest distance between leftand right medial margin of bony nostrils; 31) Interorbitwidth (IO), shortest distance between left and right medialmargin of bony orbit; 32) Nostril orbit length (NE), shortestdistance between posterior margin of left bony nostrils toanterior margin of left bony orbit; 33) Bony orbit length (E),maximum distance between anteriormost margin of bonyorbit to posteriormost margin of bony orbit; 34) Premaxillalength (PML), from medial margin of premaxilla to its lateralmargin; 35) Dentary length (DnL), from medial margin ofdentary to its lateral margin.

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