Identification of Larval Moxostoma (Catostomidae) from the Oconee River, Georgia by J. Stuart Carlton (Under the Direction of Ce

Home , Black redhorse, Catostomidae, Golden redhorse, Greater redhorse, Notchlip redhorse, River redhorse

Identification of Larval Moxostoma (Catostomidae) from the Oconee River, Georgia

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 Identification of Larval Moxostoma (Catostomidae) from the Oconee River, Georgia

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

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

ListofFigures ...... vi

ListofTables ...... vii

Chapter

1 Introduction ...... 1

2 LiteratureReview...... 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– November1996 ...... 4 1.2 Length-frequency distribution for larval Moxostoma collected April– October1999 ...... 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 notchlipandrobustredhorse...... 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 assumed to 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 in the 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 (not to 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 larval notchlip redhorse (Moxostoma collapsum) and robust redhorse (M. robustum).

Figure 4.2: Pre-anal length (expressed as % total length) in laboratory-reared larval notchlip redhorse (Moxostoma collapsum) and robust redhorse (M. robustum). 22

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

Figure 4.4: Greatest body depth (expressed as % total length) in laboratory-reared larval notchlip redhorse (Moxostoma collapsum) and robust redhorse (M. robustum). 23

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

Figure 4.6: Eye diameter (expressed as % total length) in laboratory-reared larval notchlip 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 is listed. A more detailed description of the traits appears in Table 4.2. Character measured Size class (mm) p-value of χ2 Head position 10 < 0.001 Head position 11 < 0.001 Notochord flexion 10 < 0.001 Notochord flexion 11 < 0.001 Eye pigment 11 0.001 Eye pigment 12 < 0.001 Myosepta pigment 11 < 0.001 Digestive tract 13 < 0.001 Dorsalfin 13 < 0.001 Yolk sac 14 < 0.001 Dorsalfinmargins 14 < 0.001 Anal fin 14 < 0.001 Anal fin 15 < 0.001 Pelvic flaps 15 0.001 Lip pigment 16 0.003 Snout 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 predictors appears in Table 4.2. The diagram continues in Figure 4.8

Figure 4.8: Continuation of the classiﬁcation tree for the identiﬁcation of larval robust redhorse (Moxostoma robustum) and notchlip redhorse (M. collapsum). Descriptions of 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 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 Head position Curved: head curved against yolk sac Lifted: head lifted away from yolk sac 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 Myosepta pigment Unpigmented: pigment absent on median myosepta Pigmented: dashed line of pigment along median myosepta Digestive tract Not functional: digestive tract development incomplete Functional: digestive tract fully developed and functional Dorsal fin Absent: dorsal fin and dorsal fin profile absent Developing: developing dorsal fin or dorsal fin profile evident Yolk sac Present: yolk sac present Absent: yolk sac completely absorbed Dorsal fin margins Undefined: margins of dorsal fin undefined Defined: anterior and posterior margins fin well-defined Anal fin Absent: anal fin absent; development has not begun Developing: anal fin development has begun Pelvic flaps Absent: pelvic fin development has not begun Flaps: pelvic flaps developing 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 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. Bibliography

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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

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

Yolkshape 10 0.730 Yolk shape 11 < 0.001 Headposition 10 0.001 Head position 11 < 0.001 Myomere development 10 0.001 Myomere development 11 0.003 Pectoralﬂaps 10 0.001

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Table B.1 — continued from previous page

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

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

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Table B.1 — continued from previous page

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

Digestive tract 13 < 0.001 Caudal fin II 13 < 0.001 Urostyle 13 0.008 Dorsalfin 13 < 0.001 Ventral finfold II 13 < 0.001 Dorsum melanophores 13 < 0.001 Dorsum melanophores 14 0.934 Dorsum melanophores 15 0.640 Dorsum melanophores 16 0.399 Chin pigment 13 0.689 Chin pigment 14 0.197 Yolk sac 14 < 0.001 Mouthposition 14 0.009 Pelvic fins 14 < 0.001 Dorsalfinmargins 14 < 0.001 Dorsalfinfold 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 MouthpositionII 15 0.020 AnalfinII 15 0.001

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Table B.1 — continued from previous page

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

Pelvic flaps 15 0.001 DorsalfinfoldII 15 0.001 VentralfinfoldIII 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 Dorsalfinprofile 20 0.157 AnalfinIII 20 0.003 Pelvic fins II 20 0.157 DorsalfinfoldIII 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

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Table B.2 — continued from previous page

Character Description

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

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Table B.2 — continued from previous page

Character Description

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

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Table B.2 — continued from previous page

Character Description

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

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Table B.2 — continued from previous page

Character Description

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

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Table B.2 — continued from previous page

Character Description

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;

63 64 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.

72 73 . M. col- Continued on next page d in Chapter ess advanced (a); or length (TL), standard characters were scored ments made on robust ameter (ED), pre-dorsal rs can be found in Table n Myomeres Pectoral flaps Notochord flexion Eye pigment ), abbreviated mr, and notchlip redhorse ( Moxostoma robustum ), abbreviated mc. Morphometrics were measured as describe redhorse ( Table E.1: Morphometric and descriptive character measure lapsum 3 (Methods) and are given inlength millimeters. (SL), They pre-anal include length total (PaL), head lengthfin (HL), length eye (PdF), and di greatest body depth (GBD).in Descriptive one of three ways: presentunmeasured or (u). more An advanced explanation (p); of absent the orB.2 descriptive l characte mc1mc2mc3 mcmc4 mcmc5 mc 9mc6 9.3 mcmc7 10.510.38.51.30.53.3 8.87.21.40.53.5 mc 9.17.61.40.53.4mc8 11.211.09.2-- mcmc9 11.311.19.31.40.63.6 mc 11.811.59.4-- mc 11.911.69.51.50.7 mc 11.911.69.51.50.73.6 12.111.910.01.50.74.1 na na na - a na a - a na - na a na na a na a a a a a a p a a a a a a a a p a a a a a p a p p p p a p a p p a p a a a a a a a a p p p p p p p mc10mc11mc12 mcmc13 mcmc14 12.212.010.11.60.7 mcmc15 12.512.210.21.70.7 mcmc16 12.712.310.31.90.8 mc mc 13 13.413.010.21.60.7 mc 12.710.31.80.83.6 13.513.210.61.80.8 3.6 13.513.210.71.90.8 4.0 na 4.3 na na 3.4 p 3.8 na p na 3.7 p na na p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Sample# Species TL SL PaL HL ED PdF GBD Yolkshape Headpositio 74 Continued on next page n Myomeres Pectoral flaps Notochord flexion Eye pigment mc17mc18mc19 mcmc20 mcmc21 13.513.110.71.80.8 mcmc22 13.713.210.81.80.8 mcmc23 13.713.210.12.10.8 mcmc24 13.713.110.12.10.8 mcmc25 13.813.410.71.80.9 mcmc26 13.813.110.22.10.9 mc 4.3mc27 13.813.010.02.30.8 mc 4.0mc28 13.913.510.61.90.8 mc na 4.5mc29 13.913.310.22.00.9 mc na 4.2mc30 13.913.210.42.10.8 mc na 4.3mc31 p mc 14 na 4.5mc32 p mc 14 na 4.6 13.510.81.80.84.3mc33 p mc 14 na 4.3 13.310.62.00.94.8mc34 14.113.811.11.90.8 p mc na 4.5 13.410.22.20.94.6mc35 14.113.310.12.61.0 p p mc na 4.6mc36 14.113.210.22.51.0 p p mc namc37 14.213.811.11.80.8 p p mc namc38 14.213.811.11.80.8 p p mc namc39 14.213.811.01.90.8 p p p mc 4.0 namc40 14.213.410.42.10.8 p p p mc 5.6 namc41 14.213.710.72.20.9 p p mc na pmc42 - 14.213.610.82.20.9 p p mc na p 4.2mc43 14.213.310.32.40.9 p p p mc p 4.2 namc44 14.213.510.52.10.9 p p p p mc na 4.0mc45 14.313.510.42.20.8 p p p mc na p 4.6mc46 14.313.210.12.10.9 p p mc na p p 4.6mc47 14.313.810.52.41.0 p p p mc na p 4.2mc48 14.313.710.62.51.0 p p p p mc p na 4.6mc49 14.413.810.72.30.9 p p p p mc na p 4.9mc50 14.413.710.72.20.9 p p p mc na p 4.7 p 14.513.910.82.40.8 p p p p mc na p 4.5 14.513.510.42.30.9 p p p p p mc na 4.7 14.614.011.02.20.9 p p p p p na p 5.0 14.614.010.82.30.9 p p p p na p 4.7 p p p p p p na p 4.4 p p p p p p na 4.7 p p p p p p na 4.8 p p p p na 4.7 p p p p p p p na 4.5 p p p p p p na p p p p p p p na p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample# from previous page Species TL SL PaL HL ED PdF GBD Yolkshape Headpositio 75 Continued on next page n Myomeres Pectoral flaps Notochord flexion Eye pigment mr1mr2mr3 mrmr4 mrmr5 mr 10mr6 mr 10mr7 10.059.7 9.67.81.30.52.7 mrmr8 9.67.91.30.62.6 10.310.08.11.20.52.7 mrmr9 10.410.18.21.30.52.8 mr 8.01.30.62.4 10.710.48.61.40.52.9 mr 10.710.38.51.40.62.8 mr 10.810.48.61.40.62.6 10.810.48.51.40.62.6 na na na na p na p p na p na p na p a na p a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p mr10mr11mr12 mrmr13 mrmr14 10.910.58.71.40.52.7 mrmr15 11.210.68.61.60.63.0 mrmr16 11.310.88.61.50.63.0 mr 11.310.88.61.60.62.9 mr 11.611.18.91.60.63.0 mr 11.611.08.91.70.63.1 11.611.08.91.70.63.1 na na na a na p na p na p na p p p p p p p p p p p p p p p p p p p p p p p p p u p p p p p p u p p p p p mc51mc52mc53 mcmc54 mcmc55 14.613.710.72.40.9 mcmc56 14.613.910.72.61.0 mcmc57 14.714.011.02.30.9 mcmc58 14.713.710.52.41.0 mcmc59 14.713.410.52.20.9 mcmc60 14.713.910.62.30.9 mc 4.8mc61 14.713.910.82.70.9 mc 5.0mc62 14.813.510.62.51.0 mc na 4.7mc63 14.913.310.52.51.0 mc na 4.9mc64 15.113.510.62.51.05.4 mc na 4.7mc65 15.613.911.02.71.15.3 p mc na 4.8mc66 p mc 16 na 5.5mc67 16.114.111.23.01.15.4 p mc na 5.0 14.111.32.91.15.5mc68 16.514.511.32.91.16.5 p mc na 4.9 17.114.811.83.11.26.2 p p mc na 17.314.711.93.01.26.3 p p mc na 20.817.513.84.11.36.4 p p - p p 21 - p p p 17.713.74.41.57.8 - p p p - p p p - p p - p p p p p - p p p - p p p p p - p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample# from previous page Species TL SL PaL HL ED PdF GBD Yolkshape Headpositio 76 Continued on next page n Myomeres Pectoral flaps Notochord flexion Eye pigment mr17mr18mr19 mrmr20 mrmr21 11.711.28.91.60.73.1 mrmr22 11.711.28.91.70.73.0 mrmr23 11.911.59.11.70.73.1 mrmr24 11.911.39.01.60.73.2 mrmr25 11.911.49.21.70.73.1 mrmr26 11.911.48.91.70.73.1 mrmr27 mr 12mr28 12.111.49.11.70.73.1 mr na 11.49.12.10.83.5mr29 12.211.59.01.70.83.3 mr namr30 12.211.79.01.80.83.3 mr namr31 12.211.59.11.80.73.4 mr p namr32 12.211.59.02.10.83.6 mr p namr33 12.211.69.22.10.83.5 mr p namr34 12.311.79.21.80.83.5 mr pmr35 12.411.89.41.70.83.3 na mr p p namr36 12.511.89.42.10.73.7 mr p p namr37 12.611.99.61.80.73.5 mr p na pmr38 mr 13 p p namr39 mr 13 p p p na 12.29.12.20.84.2mr40 13.112.49.62.00.83.7 mr p p p na 12.29.32.10.94.0mr41 13.312.89.62.00.84.3 mr p p namr42 p 13.312.69.82.20.84.0 mr p p p namr43 13.312.69.42.10.94.2 mr p p p p namr44 13.412.69.22.00.84.0 mr p p p p namr45 13.613.19.82.10.84.4 mr p p pmr46 p 13.713.19.62.20.94.2 na mr p p p pmr47 13.913.09.82.10.94.0 na mr p p p p namr48 mr 14 p p p p na pmr49 14.112.69.52.61.05.2 mr p p u na 13.09.72.31.05.2 pmr50 p 14.313.610.12.31.0 mr p p p p p na 14.312.89.72.51.05.3 mr p p p p p na 14.413.610.32.40.9 mr p p p p na p 14.413.710.12.30.9 p p p p p na p 14.713.710.32.30.9 p p p p p na p p p p p p p 4.5 na p p p p p na p na p p p p p 4.6 p p p p p p na 4.4 p na p p p p p 5.6 p p na p p p p p na p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample# from previous page Species TL SL PaL HL ED PdF GBD Yolkshape Headpositio 77 Continued on next page n Myomeres Pectoral flaps Notochord flexion Eye pigment mr51mr52mr53 mrmr54 mrmr55 14.812.810.02.81.0 mrmr56 14.913.310.12.61.0 mrmr57 14.913.410.12.61.0 mrmr58 mr 15mr59 mr 15 13.310.12.81.05.6mr60 15.213.410.22.71.1 mr 5.5 12.99.92.71.05.7mr61 15.313.410.32.71.1 mr 5.5mr62 15.413.210.12.81.1 mr na 5.4mr63 15.513.510.23.01.1 mr namr64 15.513.710.52.71.0 mr namr65 15.713.510.32.81.1 p mr 5.8 2.0mr66 15.713.910.62.91.1 p mr 5.7mr67 2.2 15.713.910.62.71.0 p 2.0 mr 5.9mr68 16.314.311.03.31.2 2.0 mr p 5.6mr69 16.313.910.83.01.1 p 2.1 mr 6.0 p pmr70 16.314.210.83.21.2 p 2.3 mr 5.8mr71 p 16.414.210.63.11.2 p 2.2 mr 5.6mr72 p 16.514.310.83.21.2 2.2 mr p 6.1mr73 p 16.714.311.03.31.2 p 2.3 mr 6.3 pmr74 p p 16.714.411.03.21.2 p 2.2 mr 6.0mr75 p p 16.714.310.83.21.2 p 2.5 mr 6.0 pmr76 p 16.814.411.13.21.2 2.3 mr p 6.1 pmr77 p 16.814.711.03.21.2 p 2.7 mr 6.3 p pmr78 p p 18.915.811.93.81.3 p 2.5 mr 6.1 pmr79 p p 18.915.811.63.91.3 p 2.5 mr 6.4mr80 p p p 19.215.812.04.01.4 2.6 mr p 6.3mr81 p p p 19.216.011.74.01.3 2.3 mr 6.3 p p p p p 19.416.311.93.91.4 2.5 mr p 6.3 p p p p 19.616.512.13.91.3 2.5 mr p 6.8 p p p p 20.116.612.23.81.5 2.5 p 7.0 p p p p 22.718.913.64.21.7 3.0 p 7.1 p p p p 3.1 7.0 p p p p p p p 3.2 7.1 p p p p p p 3.3 7.4 p p p p p p 3.4 7.2 p p p p p 3.3 p 8.1 p p p p p 3.2 p p p p p p p 4.0 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample# from previous page Species TL SL PaL HL ED PdF GBD Yolkshape Headpositio 78 e pigment Continued on next page finfold Eye pigment II Head pigment Myosepta pigment Peduncl mc1mcamc2mcamc3mcamc4mcpmc5mcpmc6mcpmc7mcpmc8mcpmc9mcp u u u a u a u a u a a a a a a a a a a a a p a a a a a a a p a p a p a p a p a p a a u a u a u p a a a a a a a a p a a a a a p mc10mcmc11mcmc12mcmc13mcmc14mcmc15mc pmc16mc pmc17mc pmc18mc pmc19mc pmc20mc a pmc21mc a pmc22mc a pmc23mc p pmc24mc p p pmc25mc p p pmc26mc p p p pmc27mc p p pmc28mc p a p pmc29mc p a p pmc30mc p a p pmc31mc p p p pmc32mc p p p p pmc33mc p p p p p p p p p p p p p a p p p p a p p p p p a p p p p p a p p p p a p p p p a p p p p a p p p p p a p p p p p a p p p p a p p p p a p p p p a p p p p a p p p p a p p p p a p p p p a p p p p a p p p a p p p a p p p a p p p a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample from # previous page Species Body pigment Pectoral fins Caudal fin Ventral 79 e pigment Continued on next page finfold Eye pigment II Head pigment Myosepta pigment Peduncl mc34mcmc35mcmc36mcmc37mcmc38mcmc39mc pmc40mc pmc41mc pmc42mc pmc43mc pmc44mc p pmc45mc p pmc46mc p pmc47mc p p pmc48mc p p pmc49mc p p pmc50mc p p pmc51mc p p pmc52mc p p p pmc53mc p p p pmc54mc p p p pmc55mc p p p pmc56mc p p p a pmc57mc p p p a pmc58mc p p p a pmc59mc p p p a pmc60mc p p p a pmc61mc p p p p a pmc62mc p p p p a pmc63mc p p p p a pmc64mc p p p p a pmc65mc p p p p a pmc66mc p p p p p a pmc67mc p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p a p p p p p a p p p p p a p p p p p a p p p p a p p p p a p p p p a p p p p a p p p a p p p a p p p a p p p a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample from # previous page Species Body pigment Pectoral fins Caudal fin Ventral 80 e pigment Continued on next page finfold Eye pigment II Head pigment Myosepta pigment Peduncl mr1mramr2mramr3mramr4mramr5mramr6mrpmr7mramr8mrpmr9mra u u u u u u u u u u u u u u a u a u a u a u a a a a a a a a a a a a a a a a a a a a u a u a u a u u u a u a u a u a a a a a a mr10mrmr11mrmr12mrmr13mrmr14mrmr15mr pmr16mr pmr17mr pmr18mr pmr19mr pmr20mr u pmr21mr p pmr22mr p pmr23mr p pmr24mr u p pmr25mr p p pmr26mr p p pmr27mr p p pmr28mr p p a pmr29mr p a p pmr30mr p p p pmr31mr p a p pmr32mr p p p pmr33mr a p p p p a p p a p a a p a p a p p p p a p p p p a p p p p p p a p p p p p p p p p a p p a p a p p a p u a p p p p p p a p p p a p p p p a p a p p a p a p p a p a p p a p p p p a p p p p a p a p p a p p p a p p p a p p p a a p p a p p p p p p p p p a p p p p p p p p p p p p p p p p p p p p p mc68mc p p p p a p p p Table E.1 — continuedSample from # previous page Species Body pigment Pectoral fins Caudal fin Ventral 81 e pigment Continued on next page finfold Eye pigment II Head pigment Myosepta pigment Peduncl mr34mrmr35mrmr36mrmr37mrmr38mrmr39mr pmr40mr pmr41mr pmr42mr pmr43mr pmr44mr p pmr45mr p pmr46mr p pmr47mr p pmr48mr p p pmr49mr p p pmr50mr p p pmr51mr p p pmr52mr p p p pmr53mr p p p pmr54mr p p p pmr55mr p p p pmr56mr p p p p amr57mr p p p p amr58mr p p p p amr59mr p p p p amr60mr p p p p amr61mr p p p p p amr62mr p p p p p amr63mr p p p p p amr64mr p p p p p amr65mr p p p p p amr66mr p p p p p p amr67mr p p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p a p p p p p a p p p p p a p p p p p a p p p p a p p p p a p p p p a p p p p a p p p a p p p a p p p a p p p a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample from # previous page Species Body pigment Pectoral fins Caudal fin Ventral 82 e pigment Continued on next page finfold Eye pigment II Head pigment Myosepta pigment Peduncl mr68mrmr69mrmr70mrmr71mrmr72mrmr73mr pmr74mr pmr75mr pmr76mr pmr77mr pmr78mr p pmr79mr p pmr80mr p pmr81mr p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p a p p p a p p a p p a p p a p p p a p p a p p a p p a p p a p p a p p a p p a p p a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample from # previous page Species Body pigment Pectoral fins Caudal fin Ventral 83 eduncle pigment II Continued on next page Caudal fin II Caudal fin III Ventral finfold II Yolk sac pigment P mc1mcamc2mcamc3mcamc4mcamc5mcamc6mcamc7mcamc8mcamc9mcp a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a mc10mcpmc11mcpmc12mcpmc13mcamc14mcamc15mcamc16mcamc17mcamc18mcamc19mca amc20mca amc21mca amc22mca pmc23mca pmc24mca p amc25mca p amc26mca p amc27mca p pmc28mca p pmc29mca a p pmc30mca a p pmc31mca a p pmc32mca p p pmc33mca p p p a p p p a p p p a p p p p p p p p p p p p a p p p p a p p p p p p p p p p p p p p p p p p p a p p p p a p p p p a p p p p a p p p p a p p p a p p p a a p p p a a p p p a a p p p p a p p p a p p p a p p p a p p p a p p a p p a p p a p p p p p p p p p p p p p p a p a a a a a a Table E.1 — continuedSample from # previous page Species Yolk depth Branchiostegals Opercular flaps 84 eduncle pigment II Continued on next page Caudal fin II Caudal fin III Ventral finfold II Yolk sac pigment P mc34mcamc35mcamc36mcamc37mcamc38mcamc39mcamc40mcamc41mcamc42mcamc43mca pmc44mca pmc45mca pmc46mca pmc47mca pmc48mca p pmc49mca p pmc50mca p pmc51mca p pmc52mca p pmc53mca p p pmc54mca p p pmc55mca p p pmc56mca p p pmc57mca p p p pmc58mca p p p pmc59mca p p p pmc60mca p p p pmc61mca p p p pmc62mca p p p p pmc63mca p p p p pmc64mca p p p p pmc65mca p p p p pmc66mca p p p p pmc67mca p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p a p p p p p p a p p p p p p a p p p p p p a p p p p p a p p p p p a p p p p p a p p p p p a p p p p p a p p p p a p p p p a p p p p a p p p p a p p p p a p p p a p p p a p p p a p p p a p p a p p a p p a p p a p p a p a p a p a p a p a p a a a a a a Table E.1 — continuedSample from # previous page Species Yolk depth Branchiostegals Opercular flaps 85 eduncle pigment II Continued on next page Caudal fin II Caudal fin III Ventral finfold II Yolk sac pigment P mr1mramr2mramr3mramr4mramr5mramr6mramr7mramr8mramr9mra a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a mr10mramr11mramr12mramr13mramr14mramr15mramr16mramr17mramr18mramr19mra amr20mra amr21mra amr22mra amr23mra amr24mra a amr25mra a amr26mra a amr27mra a amr28mra a amr29mra a a amr30mra a a amr31mra a a amr32mra a a amr33mra a p a a a p a a a a a a a p a a a p p a a p a a a a p p a a a p a a a a a a a a p p p a a a p a a p a p a a a a p a a a p p p a a p p a a a p p a p a p a a p a p a a a p p a a p a p a a a p a a a a a a a p p a a p p a a a a a p p a a a p p p a p a a a a p a a a p a a a a a a a a a mc68mca p p p p p p a Table E.1 — continuedSample from # previous page Species Yolk depth Branchiostegals Opercular flaps 86 eduncle pigment II Continued on next page Caudal fin II Caudal fin III Ventral finfold II Yolk sac pigment P mr34mramr35mramr36mramr37mramr38mramr39mramr40mramr41mramr42mramr43mra pmr44mra pmr45mra pmr46mra pmr47mra pmr48mra p pmr49mra p pmr50mra p pmr51mra p pmr52mra p pmr53mra p p pmr54mra p p pmr55mra p p pmr56mra p p pmr57mra p p p pmr58mra p p p pmr59mra p p p pmr60mra p p p pmr61mra p p p pmr62mra p p p p pmr63mra p p p p pmr64mra p p p p pmr65mra p p p p pmr66mra p p p p pmr67mra p p p p p p p p p p p p p p p p p a p p p p p p p p p p p a p p p p p a p p p p p a p p p p p p a p p p p p p a a p p p p p a p p p p p a p p p p p a p p p p p a p p p p p a p p p p p a p p p p a p p p p a p p p p a p p p p a p p p p a p p p a p p p a p p p a p p p a p p a p p a p p a p p a p p a p a p a p a p a p a p a a a a a a Table E.1 — continuedSample from # previous page Species Yolk depth Branchiostegals Opercular flaps 87 eduncle pigment II Continued on next page Caudal fin II Caudal fin III Ventral finfold II Yolk sac pigment P mr68mramr69mramr70mramr71mramr72mramr73mramr74mramr75mramr76mramr77mra pmr78mra pmr79mra pmr80mra pmr81mra p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p a p p a p p a p a p a p a p a p a p a a a a a a Table E.1 — continuedSample from # previous page Species Yolk depth Branchiostegals Opercular flaps 88 Continued on next page n IV Urostyle Dorsal fin Ventral finfold III Dorsum pigment mc1mcmc2mcmc3mcmc4mcmc5mcmc6mc amc7mc amc8mc amc9mc a a a a a a a a a a a a a a a a a a a a u a u a u a u a u u u u u u u a u u a u u a u a u a a u a a a a a a a a a a a a a a a a a a a a a mc10mcmc11mcmc12mcmc13mcmc14mcmc15mc amc16mc amc17mc amc18mc amc19mc a amc20mc a amc21mc a amc22mc a a amc23mc a a amc24mc a a amc25mc a a amc26mc a a amc27mc a u a amc28mc a u a amc29mc a u a amc30mc a a a amc31mc u a a a amc32mc u a a a pmc33mc u a a a p a a a a a p a a a a a p p a a p a p p a p a a p p a a a a p a p a p p p a p a p a p a p a p a p a p a p a p a a p a p a a p a a p a a p a a p a a p a a a a p a a a p p a p p p p a a a p p a a a p p a a p p p a p p p a p p a a p a p p p p p p p p p a p a p a p a p p p p p Table E.1 — continuedSample from # previous page Species Yolk depth II Nares Digestive tract Caudal fi 89 Continued on next page n IV Urostyle Dorsal fin Ventral finfold III Dorsum pigment mc34mcmc35mcmc36mcmc37mcmc38mcmc39mc pmc40mc pmc41mc pmc42mc p pmc43mc p pmc44mc p pmc45mc p pmc46mc p p pmc47mc p p pmc48mc p p pmc49mc p p pmc50mc p p pmc51mc p p p pmc52mc p p p pmc53mc p p p pmc54mc p p p p pmc55mc p p p p pmc56mc p p p p pmc57mc p p p p pmc58mc p p p p p pmc59mc p p p p p pmc60mc p p p p p pmc61mc p p p p p pmc62mc p p p p p p pmc63mc p p p p a p pmc64mc p p p p p p pmc65mc p p p p p p pmc66mc p p p p p p pmc67mc p p p p a p p p p a p p p p p p p a p p a p p p p p p p p p p p p p p p a p p p p p p p p p p p p p p p p p p p a p p p p p p a p p p p p p p p a p p p p p p p p p a p a p p p p p p p p p p p p p p a p p p p p p p p p a a p p p p p p p p p p p p p p p p p p p p p p p p p a p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample from # previous page Species Yolk depth II Nares Digestive tract Caudal fi 90 Continued on next page n IV Urostyle Dorsal fin Ventral finfold III Dorsum pigment mr1mrmr2mrmr3mrmr4mrmr5mrmr6mr amr7mr amr8mr amr9mr a a a a a a a a a a a a a a a a a a a a u a u a u a u a u u u u u u u a u u a u u a u a u a a u a a a a a a a a a a a a a a a a a a a a a mr10mrmr11mrmr12mrmr13mrmr14mrmr15mr amr16mr amr17mr amr18mr amr19mr a amr20mr a amr21mr a amr22mr a a amr23mr a a amr24mr a a amr25mr a a amr26mr a a amr27mr a u a amr28mr a u a amr29mr a u a amr30mr a u a amr31mr u a u a amr32mr u a u a amr33mr u a u a a u a a u a a u a a u a a u a a u a a u a a u a a u a a u a a a u a a u a a u a a u a a u a a u a a u a a u a a u a u a a u a u a a a u a u a a a u a u a a u a u a a u a u a a u a u a a u a u a a u a a a u a a a u a a a u a a a a a a a a a a a a a a a a a a a a a a a a a a mc68mc p p p p p p p p Table E.1 — continuedSample from # previous page Species Yolk depth II Nares Digestive tract Caudal fi 91 Continued on next page n IV Urostyle Dorsal fin Ventral finfold III Dorsum pigment mr34mrmr35mrmr36mrmr37mrmr38mrmr39mr pmr40mr pmr41mr amr42mr amr43mr p pmr44mr p amr45mr p amr46mr p a pmr47mr p p pmr48mr p a pmr49mr p a pmr50mr p a pmr51mr p p a pmr52mr p p a pmr53mr p p p pmr54mr p p p pmr55mr p p p a p pmr56mr p p p pmr57mr p p p p pmr58mr p p p p p pmr59mr p p p p p pmr60mr p p p p p pmr61mr p p p a p p pmr62mr p p p p p a pmr63mr p p p p p pmr64mr p p p p p p a pmr65mr p p p p p p pmr66mr p p p p p p pmr67mr p p p p p p p p p p p p p p p p p a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p a p p p p p p p p p p p p p p a p p p p p p p p a p p p p p p p a p p p p p p p p p a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p a p p p p p p p p Table E.1 — continuedSample from # previous page Species Yolk depth II Nares Digestive tract Caudal fi 92 Continued on next page n IV Urostyle Dorsal fin Ventral finfold III Dorsum pigment mr68mrmr69mrmr70mrmr71mrmr72mrmr73mr pmr74mr pmr75mr pmr76mr pmr77mr p pmr78mr p pmr79mr p pmr80mr p p pmr81mr p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p a p p p a p p p a p p a p p p p p a p p p a p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample from # previous page Species Yolk depth II Nares Digestive tract Caudal fi 93 ment Continued on next page fins Dorsal fin margins Dorsal finfold Anal fin Arrow-shaped pig mc1mcmc2mcmc3mcmc4mcmc5mcmc6mc pmc7mc pmc8mc pmc9mc p a p a p a p a p a u p a u a u a u a u a u a u a u a u a u a u a u a u a u u u u u u u u u a u a u a u a u a u a a a a a a a a a a a a a mc10mcmc11mcmc12mcmc13mcmc14mcmc15mc pmc16mc pmc17mc pmc18mc pmc19mc a pmc20mc a pmc21mc a pmc22mc a pmc23mc a u pmc24mc a u pmc25mc a u pmc26mc a u pmc27mc a u p amc28mc a u p amc29mc a u p amc30mc a u p amc31mc a u p amc32mc u a u p amc33mc u a u p a u a u p a u a u p a u a u p a u a u u p a u a u u p a u a u u a u a a u a a u a a u a a u a a u a a u a u a a u a u a a u a u a a a u a u a a a u u a a a u u a a a a u a a a a u a a a a u a a a u a a a u a a a a a a a a a a a a a a a p a a a a a p a a p a p a p p p p p p p p Table E.1 — continuedSample from # previous page Species Chin pigment Yolk sac Mouth position Pelvic 94 ment Continued on next page fins Dorsal fin margins Dorsal finfold Anal fin Arrow-shaped pig mc34mcmc35mcmc36mcmc37mcmc38mcmc39mc pmc40mc pmc41mc pmc42mc pmc43mc a pmc44mc a pmc45mc a pmc46mc a pmc47mc a a pmc48mc a a pmc49mc a a pmc50mc a a pmc51mc a a p amc52mc a a p amc53mc a a p amc54mc p a p amc55mc a a p amc56mc a a a a amc57mc a a a p amc58mc a a a p amc59mc a a a a amc60mc a p a p amc61mc p a a a p amc62mc a a a a p amc63mc p a a a a amc64mc p a a a p amc65mc p a a a a a amc66mc a a a a a a amc67mc p a a p a a a p a a a a a a p a p a a a a p p a p a a a a p p a p a a a a p a p a a a a a p p p p a a a p p a p p a a p p p p a a a p p p p a a a p p a p a p p p p a a p p p p a a p p p p a a p p p a a p p p p a p p p p a p p p p a p a p p a p p p a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample from # previous page Species Chin pigment Yolk sac Mouth position Pelvic 95 ment Continued on next page fins Dorsal fin margins Dorsal finfold Anal fin Arrow-shaped pig mr1mrmr2mrmr3mrmr4mrmr5mrmr6mr pmr7mr pmr8mr pmr9mr p a p a p a p a p a u p a u a u a u a u a u a u a u a u a u a u a u a u a u u u u u u u u u a u a u a u a u a u a a a a a a a a a a a a a mr10mrmr11mrmr12mrmr13mrmr14mrmr15mr pmr16mr pmr17mr pmr18mr pmr19mr a pmr20mr a pmr21mr a pmr22mr a pmr23mr a u pmr24mr a u pmr25mr a u pmr26mr a u pmr27mr a u p amr28mr a u p amr29mr a u p amr30mr a u p amr31mr a u p amr32mr u a u p amr33mr u a u p a u a u p a u a u p a u a u p a u a u u p a u a u u p a u a u u a u a u u a a u a u u a a u a u u a a u u u a a u u u a a u u u a a a u u u a a a u u a a a u u a a a u u a a a u u a a a u u a a u u a a u u a a u u a a u u a a u a a u a a u a a u a a u a a a a a a a a a a a a a mc68mc a p p p p p p p Table E.1 — continuedSample from # previous page Species Chin pigment Yolk sac Mouth position Pelvic 96 ment Continued on next page fins Dorsal fin margins Dorsal finfold Anal fin Arrow-shaped pig mr34mrmr35mrmr36mrmr37mrmr38mrmr39mr pmr40mr pmr41mr pmr42mr pmr43mr a pmr44mr a pmr45mr a pmr46mr a pmr47mr a u pmr48mr a u pmr49mr a u amr50mr a u amr51mr a u a amr52mr a u a amr53mr u p a amr54mr u p a amr55mr u a a amr56mr u u a a amr57mr u p a a amr58mr u p a a amr59mr u p a a amr60mr u a a a amr61mr u a p u a pmr62mr u p p u p amr63mr u p p u a pmr64mr u p a u a pmr65mr a u p p u a pmr66mr a p p p u a pmr67mr a p p p u p a a p p p u a p a p p p u a p a a p p p u a p a a p p p p p a a a p p p p p a a a p p p p p a a p p p p p p a p p p p p a a p p p p p a a p p p p a a p p p p a a p p p p p p p p p p p p p p p a a p p p p a p p p p a p p p p a p p p a p p p a p p p p p p p p p p p a p p p p p p p p a p p p p p p p p p p p p p p p p p Table E.1 — continuedSample from # previous page Species Chin pigment Yolk sac Mouth position Pelvic 97 ment Continued on next page fins Dorsal fin margins Dorsal finfold Anal fin Arrow-shaped pig mr68mrmr69mrmr70mrmr71mrmr72mrmr73mr amr74mr amr75mr amr76mr amr77mr p amr78mr p amr79mr p amr80mr p amr81mr p p a p p a p p a p p a p p a p p p p a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p a p p a p p a p p a p p a p a p p p a p p p p p Table E.1 — continuedSample from # previous page Species Chin pigment Yolk sac Mouth position Pelvic 98 entral finfold IV Continued on next page ent Mouth position II Anal fin II Pelvic flaps Dorsal finfold II V mc1mcmc2mcmc3mcmc4mcmc5mcmc6mcmc7mc amc8mc amc9mc a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a u a u a u a u a u u u u u u u u u u u u u u mc10mcmc11mcmc12mcmc13mcmc14mcmc15mcmc16mc amc17mc amc18mc amc19mc amc20mc amc21mc a amc22mc a amc23mc a amc24mc a amc25mc a amc26mc a a amc27mc a a amc28mc a a amc29mc a a amc30mc a a amc31mc a a a amc32mc a a a amc33mc a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a u a a a a u a a a a u a a a a u a a a a u a a a a u a u a a u a u a a u a u a a u a u a a u a u a a u a u a a u u a a u u a a u u a a u u a a u u a u u a a u a a u a a u a u a u a a a a a a a a a Table E.1 — continuedSample from # previous page Species Gut melanophores Jaw pigment Gill arch pigm 99 entral finfold IV Continued on next page ent Mouth position II Anal fin II Pelvic flaps Dorsal finfold II V mc34mcmc35mcmc36mcmc37mcmc38mcmc39mcmc40mc amc41mc amc42mc amc43mc amc44mc amc45mc a amc46mc a amc47mc a amc48mc a amc49mc p amc50mc a a amc51mc a a amc52mc a a amc53mc a a amc54mc a a amc55mc a a a amc56mc a p a amc57mc a a a amc58mc a a a amc59mc a a a amc60mc a a a a amc61mc a a a a amc62mc a a a a amc63mc a a a a amc64mc a a a a a amc65mc a a a a a amc66mc a a a a a amc67mc a a a a a a a a a a a a a a a a a a a a a a a a a a a a a p a a a a a a p a a a a a a p a a a a a p a a a a a a p a a a a a a p a a a a a p p a a a a a p p a a a a a p a a a a a p a a a a a p a a a a a p a a a a a p a a a p a a a a p a a a a p a a a a p a a a a p a a a p p a a a p a a a p a a a p a p a p a p a p a p a a p a a p a p p a p a p a p a p a p a a p p a a Table E.1 — continuedSample from # previous page Species Gut melanophores Jaw pigment Gill arch pigm 100 entral finfold IV Continued on next page ent Mouth position II Anal fin II Pelvic flaps Dorsal finfold II V mr1mrmr2mrmr3mrmr4mrmr5mrmr6mrmr7mr amr8mr amr9mr a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a u a u a u a u a u u u u u u u u u u u u u u mr10mrmr11mrmr12mrmr13mrmr14mrmr15mrmr16mr amr17mr amr18mr amr19mr amr20mr amr21mr a amr22mr a amr23mr a amr24mr a amr25mr a amr26mr a a amr27mr a a amr28mr a a amr29mr a a amr30mr a a amr31mr a a a amr32mr a a a amr33mr a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a u a a a a u a a a a u a a a a u a a a a u a a a a u a u a a u a u a a u a u a a u a u a a u a u a a u a u a a u u a a u u a a u u a a u u a a u u a u u a u u a u u a u u u u u u u u u u u u u u u mc68mc a p p p p p p a Table E.1 — continuedSample from # previous page Species Gut melanophores Jaw pigment Gill arch pigm 101 entral finfold IV Continued on next page ent Mouth position II Anal fin II Pelvic flaps Dorsal finfold II V mr34mrmr35mrmr36mrmr37mrmr38mrmr39mrmr40mr amr41mr amr42mr amr43mr amr44mr amr45mr a amr46mr a amr47mr a amr48mr a amr49mr a amr50mr a a amr51mr a p amr52mr a a amr53mr a a amr54mr a a amr55mr a a p amr56mr a p a amr57mr a a p amr58mr a a p amr59mr a a p amr60mr a a p a amr61mr a a p a amr62mr a a p a amr63mr a a p a amr64mr a a p a a amr65mr a a p a a amr66mr a a p a a amr67mr a a p a a a a a p a a a u a a p a a a u p a p a a a u p a p a a a u p a p a a a u p a p a a a u p u p a a a u p u a p a p u p u p a a p u p u p a a p u p u p a a p a p u a a p a p u a p p a p u a p p a p u a p p a p u a p p a p a p p a p a p p a p a p p a p a p p a p a p p p p a p p p a p p p a p p p a p p p a p p a p p a p p a p p a p p a p a p a p a p a a a p p a Table E.1 — continuedSample from # previous page Species Gut melanophores Jaw pigment Gill arch pigm 102 entral finfold IV Continued on next page ent Mouth position II Anal fin II Pelvic flaps Dorsal finfold II V mr68mrmr69mrmr70mrmr71mrmr72mrmr73mrmr74mr amr75mr amr76mr amr77mr amr78mr amr79mr a pmr80mr a pmr81mr a p a p a p p a p p a p p a p p a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p Table E.1 — continuedSample from # previous page Species Gut melanophores Jaw pigment Gill arch pigm 103 III Continued on next page ut pigment Jaw pigment II Head profile Dorsal fin profile Anal fin mc1mcmc2mcmc3mcmc4mcmc5mcmc6mcmc7mc umc8mc umc9mc u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u a u a u a u a u a u a u a u a u a u u u u u u u u u u u u u u mc10mcmc11mcmc12mcmc13mcmc14mcmc15mcmc16mc umc17mc umc18mc umc19mc umc20mc u umc21mc u umc22mc u umc23mc u umc24mc u u umc25mc u u umc26mc u u umc27mc u u umc28mc u u umc29mc u u u umc30mc u u u umc31mc u u u umc32mc u u u umc33mc u u u u u u u u u u u u u u u u u u u u u u u u u u u a u u u u u a u u u u a u u u u a u u u u a u u u u u a u u u u u a u u u u a u u u u a u u u u a u u u u u a u u u u a u u u u a u u u u a u u u u a u u u a u u u a u u u a u u u a u u u a u u a u u a u u a u u a u u u u u u u u u u u u u u u Table E.1 — continuedSample# from previous page Species Operculumlength Pelvic fins Lippigment Sno 104 III Continued on next page ut pigment Jaw pigment II Head profile Dorsal fin profile Anal fin mc34mcmc35mcmc36mcmc37mcmc38mcmc39mcmc40mc umc41mc umc42mc umc43mc umc44mc u umc45mc u umc46mc u umc47mc u umc48mc u u umc49mc u u umc50mc u u umc51mc u u umc52mc u u umc53mc u u u umc54mc u u u umc55mc u u u umc56mc u u u umc57mc u u u u umc58mc u u u u umc59mc u u u u umc60mc u u u u umc61mc u u u u umc62mc u u a u u umc63mc u u a u u umc64mc u u a u u umc65mc u u a u u umc66mc u u a u u umc67mc u u u a u u u u u a a u u u u u a a u u u u u a a u u u u u p a u u u u u u p a u u p u u u p a u u p u u u a u u p u u u a u u p u u u a a u p u u u a a u p u u u a a u u u u a a u u u u a a u u u a a a u u a u a u u a u a u u a u a u u a u a u u a u a u u u a u u u a u a u a u a u a u p u a u u a u u a u u a u u a u u u u u a u a u a u u a a p Table E.1 — continuedSample# from previous page Species Operculumlength Pelvic fins Lippigment Sno 105 III Continued on next page ut pigment Jaw pigment II Head profile Dorsal fin profile Anal fin mr1mrmr2mrmr3mrmr4mrmr5mrmr6mrmr7mr umr8mr umr9mr u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u a u a u a u a u a u a u a u a u a u u u u u u u u u u u u u u mr10mrmr11mrmr12mrmr13mrmr14mrmr15mrmr16mr umr17mr umr18mr umr19mr umr20mr u umr21mr u umr22mr u umr23mr u umr24mr u u umr25mr u u umr26mr u u umr27mr u u umr28mr u u umr29mr u u u umr30mr u u u umr31mr u u u umr32mr u u u umr33mr u u u u u u u u u u u u u u u u u u u u u u u u u u u a u u u u u a u u u u a u u u u a u u u u a u u u u u a u u u u u a u u u u a u u u u a u u u u a u u u u u a u u u u a u u u u a u u u u a u u u u a u u u a u u u a u u u a u u u a u u u a u u a u u a u u a u u a u u u u u u u u u u u u u u u mc68mc p p a a p a a p Table E.1 — continuedSample# from previous page Species Operculumlength Pelvic fins Lippigment Sno 106 III Continued on next page ut pigment Jaw pigment II Head profile Dorsal fin profile Anal fin mr34mrmr35mrmr36mrmr37mrmr38mrmr39mrmr40mr umr41mr umr42mr umr43mr umr44mr u umr45mr u umr46mr u umr47mr u umr48mr u u umr49mr u u umr50mr u u umr51mr u u umr52mr u u umr53mr u u u umr54mr u u u umr55mr u u u umr56mr u u u umr57mr u u u umr58mr u u u u umr59mr u u u u umr60mr u u u u umr61mr u u u u umr62mr u u u a u umr63mr u u u a u umr64mr u u u a u amr65mr u u u a u amr66mr u u u a u amr67mr u u u u a u a u u u u a u a u u u u a u a u u u p a u a u u u p a u u a u u u p a u u a u u u a a u u a u u u a u u p u u u a u u p u u u a u u p u u u a u u p u u u a a u u u u a a u u u u a a u u u u a a u u u u a u u a u a u u a u a u u a u a u u a u a u u u a u u u a u u u a u u u a u u u a u u a u u a u u a u u a u u u u u u u u u u u u u u u Table E.1 — continuedSample# from previous page Species Operculumlength Pelvic fins Lippigment Sno 107 III Continued on next page ut pigment Jaw pigment II Head profile Dorsal fin profile Anal fin mr68mrmr69mrmr70mrmr71mrmr72mrmr73mrmr74mr pmr75mr pmr76mr pmr77mr pmr78mr p pmr79mr p pmr80mr p pmr81mr p p p a p p a p p a p p a p p a p a p a p a a p a a p a a p a a p u a a u a a u a a u a a u a a u a a p a a a a a p a a p u a p u p p u p p u p p u p u u p u p p u p p u p p u p u p p p p p p p p p p p p Table E.1 — continuedSample# from previous page Species Operculumlength Pelvic fins Lippigment Sno 108 cale pigment Dorsum pigment II Continued on next page mc1mcmc2mcmc3mcmc4mcmc5mcmc6mc umc7mc umc8mc umc9mc u u u u u u u u u u u a u a u a u a u a u a u a u a u a u u u u u u u u u u u u u u mc10mcmc11mcmc12mcmc13mcmc14mcmc15mc umc16mc umc17mc umc18mc umc19mc umc20mc u umc21mc u umc22mc u umc23mc u umc24mc u umc25mc a u umc26mc a u umc27mc a u umc28mc a u umc29mc a u u umc30mc a u u umc31mc a u u umc32mc a u u umc33mc a u u u a u u u u a u u u u a u u u u a u u u u a u u u u a u u u a u u u a u u u a u u u a u u u a u u a u u a u u a u u a u u u u u u u u u u u u u u u Table E.1 — continuedSample from # previous page Species Pelvic fins II Dorsal finfold III Squamation S 109 cale pigment Dorsum pigment II Continued on next page mc34mcmc35mcmc36mcmc37mcmc38mcmc39mc umc40mc umc41mc umc42mc umc43mc umc44mc u umc45mc u umc46mc u umc47mc u umc48mc u umc49mc a u umc50mc a u umc51mc a u umc52mc a u umc53mc a u u umc54mc a u u umc55mc a u u umc56mc a u u umc57mc a u u umc58mc a u u u u mc59mc a u u u u mc60mc a u u u u mc61mc a u u u u mc62mc a u u u u mc63mc a u u u u mc64mc a u u u u mc65mc a u u u u mc66mc a u u u u mc67mc a u u u u a u u u u a u u u u a u u a u a u u p u a u u a u a u u u a u u u a u p u a u a u a u p u a u u a u u a u u a u u a u u u u u u u u p u u u u u p Table E.1 — continuedSample from # previous page Species Pelvic fins II Dorsal finfold III Squamation S 110 cale pigment Dorsum pigment II Continued on next page mr1mrmr2mrmr3mrmr4mrmr5mrmr6mr umr7mr umr8mr umr9mr u u u u u u u u u u u a u a u a u a u a a a a a a a a a a a u a u a u a u u u u u u mr10mrmr11mrmr12mrmr13mrmr14mrmr15mr umr16mr umr17mr umr18mr umr19mr umr20mr u umr21mr u umr22mr u umr23mr u umr24mr u umr25mr a u umr26mr a u umr27mr a u umr28mr a u umr29mr a u a umr30mr a u a umr31mr a u a umr32mr a u a umr33mr a u a u a u a u u a u a u u a u a u u a u a u u a u a u u a u a u a u a u a u a u a u a u a u a u a a u a a u a a u a a u a a u a u a u a u a u u u u u u mc68mc p a a a p Table E.1 — continuedSample from # previous page Species Pelvic fins II Dorsal finfold III Squamation S 111 cale pigment Dorsum pigment II Continued on next page mr34mrmr35mrmr36mrmr37mrmr38mrmr39mr umr40mr umr41mr umr42mr umr43mr umr44mr u umr45mr u umr46mr u umr47mr u umr48mr u umr49mr a u umr50mr a u umr51mr a u umr52mr a u umr53mr a u a umr54mr a a u umr55mr a u a umr56mr a u a umr57mr a u a umr58mr a u a u u mr59mr a u a u u mr60mr a u a u u mr61mr a u a u u mr62mr a u a u u mr63mr a u a u u mr64mr a u a u u mr65mr a u a u u mr66mr a u a u u mr67mr a u a u u a u a u u a u a u u a u a u u a u a u u a u a u u a u a u a u a u a u a u a u a u a u a u a a u a a u a a u a a u a a u a u a u a u a u u u u u u Table E.1 — continuedSample from # previous page Species Pelvic fins II Dorsal finfold III Squamation S 112 cale pigment Dorsum pigment II Continued on next page mr68mrmr69mrmr70mrmr71mrmr72mrmr73mr umr74mr umr75mr umr76mr umr77mr umr78mr u umr79mr u pmr80mr u pmr81mr u p u p a u p a p p a p p a p p a p a a a p a p a a p a a p a a a u a a u a a u p a u p a u a u a u p u p u u u u p p Table E.1 — continuedSample from # previous page Species Pelvic fins II Dorsal finfold III Squamation S