Decoupling of Molecular and Morphological Evolution in Deep Lineages of a Meiobenthic Harpacticoid Copepod

Axayácatl Rocha-Olivares, John W. Fleeger and David W. Foltz

Department of Biological Sciences, Louisiana State University


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
Molecular and biochemical genetic analyses have revealed that many marine invertebrate taxa, including some well-studied and presumably cosmopolitan species, are actually complexes of sibling species. When morphological differences are slight and estimated divergence times are old, data suggest either unusually high rates of sequence evolution or long-term morphological stasis. Here, five gene regions (mitochondrial cytochrome oxidase subunit I and large-subunit ribosomal 16S rDNA and nuclear ITS1, 5.8S rDNA, and ITS2) were analyzed in four geographic samples of the meiobenthic harpacticoid copepod Cletocamptus deitersi. Molecular sequences revealed four extremely differentiated molecular lineages with unalignable nuclear intergenic spacers and mitochondrial uncorrected divergences reaching 25% (cytochrome oxidase) and 36% (16S rDNA). These levels of divergence are greater than those reported previously for congeneric species in diverse invertebrate taxa, including crustaceans. The nominally intraspecific divergence matches or exceeds the corresponding divergence from a known congener (Cletocamptus helobius). A molecular clock applied to the cytochrome oxidase subunit I data suggests that these lineages split in the Miocene, consistent with the fossil record of a North American Cletocamptus from the same period. Morphological differences among the major lineages are subtle but congruent with the patterns of genetic differentiation. Our conclusion, based on concordant patterns of variation in two mitochondrial and three nuclear gene regions, as well as morphological observations, is that C. deitersi in North America is composed of at least four separate species by the genealogical concordance, phylogenetic, and morphological-species criteria. Alternative explanations for the deep phylogenetic nodes and apparent morphological stasis, including high rates of sequence evolution, balancing selection, and genetic signatures of historical events, are considered unlikely.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
When morphological stasis persists after speciation events, resulting species may continue to diverge genetically in the absence of morphological differentiation, producing cryptic species. An inability to recognize cryptic or sibling species using traditional morphological analyses has hindered our understanding of ecological and evolutionary processes, with negative consequences such as underestimation of true levels of species-richness, overestimation of potential for long-distance dispersal in putatively cosmopolitan species, failure to recognize cryptic biological invasions, and misinterpretation of ecological and paleoecological data (e.g., Knowlton et al. 1992Citation ; Geller et al. 1997Citation ).

Cryptic species of small metazoan invertebrates (<2 mm body length and <5 µg dry weight) are particularly difficult to identify using morphological criteria alone. Small body size generally reduces the number of taxonomic characters available, and many groups have soft bodies that preserve poorly. With improved microscopic techniques and more consistent use of an advanced conceptual framework of systematics methods, many invertebrates once supposed cosmopolitan are now considered to have more limited geographic ranges. For example, the fraction of cyclopoid copepods that are putatively Holarctic has decreased from 68% to 28% as the proportion of Nearctic species shared with the Palearctic region has steadily declined (Reid 1998Citation ). Cryptic species are also known to occur among harpacticoid copepods (e.g., Ganz and Burton 1995Citation ); however, improved taxonomic techniques and inclusion of characters such as body ornamentation suggest that harpacticoid cryptic species can frequently be distinguished based on subtle morphological characters (Huys et al. 1996Citation ), thereby representing "pseudosibling species" sensu Knowlton (1993)Citation .

Cletocamptus deitersi (Richard 1897) is a canthocamptid meiobenthic copepod with a highly cosmopolitan distribution reported from inland brines (seeps, streams, and lakes), as well as coastal estuaries and mangroves on all continents except Europe (Mielke 2000)Citation . This species has been shown to be morphologically polymorphic within and between populations (e.g., Fleeger 1980Citation ; Mielke 2000Citation ). Cletocamptus deitersi lacks a planktonic dispersing larval stage, since free-living larval stages develop benthically, and adults and juveniles are capable of only short-distance (meter-scale) dispersal through the water column (Sun and Fleeger 1994Citation ). The geographical isolation of inland brines suggests that long-distance migration and colonization events are rare and that gene flow should be limited among inland populations. Morphological variability and the potential for geographic differentiation make C. deitersi a good candidate for analyses of geographic genetic variation aimed at determining the existence of cryptic species and understanding how rates of morphological and molecular evolution are related.

Using a multilocus approach, we analyzed Cletocamptus deitersi from four localities in North America to determine if separate populations were represented by morphologically and/or genetically differentiated species. We also obtained genetic data from Cletocamptus helobius, a readily identifiable and morphologically differentiated congeneric species, to establish a reference level of indisputably interspecific genetic differentiation. An undescribed harpacticoid (Canuellidae: Coullana sp.) and a planktonic calanoid (Calanus pacificus Brodsky 1948) were used as outgroups in phylogenetic reconstructions.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
Copepod Samples and DNA Extraction
Samples were collected at an inland brine seep in Alabama (June 1998, 31°23'N, 87°53'W) and at two locations on the coast of Louisiana (Port Fourchon, December 1998, 29°05.4'N 90°05.8'W, and Cocodrie, January 1999, 29°15.2'N, 90°39.8'W). Cletocamptus deitersi was also obtained from laboratory cultures established from collections made in the Salton Sea, California (July 1997, 33°13'N, 115°52'W) and at the mouth of the Estero del Yugo estuary (January 2000, 23°18.14'N, 106°29'W) in Mazatlán, Sinaloa, Mexico. These environments are very different. For example, salinity at the Alabama brine seep reaches 45 ppt close to the source, and the stream is located in a bottomland forest subject to annual flood and drought cycles some 83 km from the coast. The Louisiana salt marsh is tidally flooded, and salinity ranges from 0 to 28 ppt. The Salton Sea, where salinity has been increasing to over 44 ppt in recent times, was created in 1905 and is more than 100 km inland from the Pacific Ocean and from the Colorado River Delta in the upper Gulf of California.

Field-collected copepods were removed from sieved sediment and fixed in 95% ethanol. All harpacticoids were carefully identified by one of us (J.W.F.). We extracted total genomic DNA from individual copepods based on Schizas et al. (1997)Citation . Ethanol-preserved specimens of C. pacificus from southern California (32°25'N, 119°58'W) were provided and identified by Annie Townsend (Scripps Institution of Oceanography). DNA extraction for these copepods was done by standard proteinase-K digestion and phenol-chloroform-isoamyl organic extractions, followed by ethanol precipitation.

PCR Amplification and Sequencing
All copepods were subject to an initial PCR amplification of the mitochondrial DNA (mtDNA) cytochrome oxidase subunit I (COX-I) gene using universal primers (Folmer et al. 1994; 0.72 mM total dNTP, 2.5 mM MgCl2, 1 U Promega Taq in manufacturer's "A" buffer; conditions for Perkin Elmer 480 thermal cycling were as follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 40°C, and 60 s at 72°C, and a final extension time of 5 min at 72°C). We sequenced two additional gene regions in copepods representative of the major lineages identified on the basis of COX-I sequences (except for the Mexican copepods, for which all gene regions were sequenced in all individuals): (1) part of the mitochondrial large-subunit ribosomal DNA (LSU rDNA 16S), amplified using Palumbi's (1996)Citation 16SAR and 16SBR universal primers, and (2) part of the nuclear rDNA, comprising all of ITS1, ITS2, and the intervening 5.8S rDNA, amplified using Heath, Rawson, and Hilbish's (1995) ITS primers (cf. table 1 in Heath, Rawson, and Hilbish 1995Citation ). We designed Cletocamptus-specific internal primers for reamplification of weak initial reactions (COX-I: CLEintF [TTTTGATTTTCTYATCCAGC] and CLEintR [CCTAGTAANGARGAAATTCC]), for amplification when universal primers failed (16S: 16SciF [YTAAGGTAGCATAGTAA] and 16SciR [TTAATTCAACATCGANGTC]), and for sequencing of long PCR products (nuclear rDNA: 5.8SciF [GGGGTCGATGAAGAACG] and 5.8SciR [CCCTGAGCCAGACATGG]). Target PCR products were electrophoresed, excised from gels (2% agarose), purified using columns (QiaQuick, Promega), and sequenced using Applied BioSystems ABI-Prism Big-Dye terminator chemistry (Perkin-Elmer) in scaled-down reactions and run on an ABI-377 Gene Analyzer. Representative nucleotide sequences have been submitted to GenBank (AF315001AF315033).


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Table 1 Number of Copepods (n), Number of Distinct mtDNA Haplotypes or Nuclear Gene Copies (h), and Accession Numbers of Representative Sequences Submitted to GenBank (GB) of Each Copepod Species Genotyped by Direct Sequencing (COX-I, 16S, n rDNA) or Multiplex Haplotype-specific PCR of the COX-I Gene (MHS-PCR)

 
Multiplex Haplotype-Specific PCR
We designed a rapid, cost-effective method of large-scale genotype screening of individual copepods, using a multiplex haplotype-specific PCR reaction (MHS-PCR; Rocha-Olivares 1998Citation ), to assign copepods to one of the first two COX-I mtDNA major lineages. Initial LCOI-HCOI amplicons were used as templates in the MHS-PCR, in which the size of the products, visualized in an agarose gel, could be used for PCR-based genotyping. Multiplexing with nested primers produced a positive control amplicon in all haplotypes and a second, haplotype-specific, amplicon, whose size would be diagnostic (fig. 1 ). All MHS-PCR batches included experimental positive and negative controls. We sequenced a number of copepods genotyped with MHS-PCR to verify the accuracy of the method.



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Fig. 1.—Multiple haplotype-specific PCR (MHS-PCR) used to genotype individual Cletocamptus deitersi based on cytochrome oxidase subunit I amplification. A, A pair of nonspecific primers is used as a positive control, and an additional pair of haplotype-specific primers yields amplicons of diagnostic size used for genotyping. B, Negative gel image of the results obtained with C. deitersi. Lanes 1 and 8 show molecular markers; lane 2 shows a type I copepod verified by direct sequencing (427- and 349-bp bands); lanes 3 and 4 show unknown copepods from Louisiana; lanes 5 and 6 show unknown copepods from Alabama; and lane 7 shows a type II copepod verified by direct sequencing (427- and 253-bp bands)

 
Data Analyses
Orthologous nucleotide sequences were aligned with the CLUSTAL W algorithm (Thompson, Higgins, and Gibson 1994Citation ) with default parameters. In mitochondrial and nuclear rDNAs, inferred indels were further adjusted by eye to minimize those separated by one or very few bases. Aligned nucleotide sequences, with the exception of internal transcribed spacer (ITS) regions, were used to reconstruct gene trees using maximum parsimony (MP) and neighbor joining (NJ) with the program PAUP*, version 4.0b4a (Swofford 1998Citation ). NJ searches were made with distances computed using a best-fit model of nucleotide substitution. Models were fit to each data set (COX-I, 16S, and 5.8S) under a maximum-likelihood framework and tested for significance (overall {alpha} = 0.05 after Bonferroni correction) with likelihood ratio tests (LRTs) using the program Modeltest, version 3.04 (Posada and Crandall 1998Citation ). Branch support was assessed by 1,000 nonparametric bootstrap replicates. Phylogenetic analyses of the mtDNA 16S rDNA were performed on the data set bounded by the 16SciF-16SciR primers (313 bp), not the entire 16SAR-16SBR alignment (466 bp), which was incomplete for the Mexican C. deitersi and C. helobius. Competing phylogenetic hypotheses and the existence of a molecular clock were tested under a likelihood framework with LRTs. The program Seq-Gen (Rambaut and Grassly 1997Citation ) was used for parametric bootstrapping of data sets. Rates of synonymous and nonsynonymous substitutions were computed following Yang and Nielsen (1998)Citation with the program PAML, version 3.0b (Yang 1997Citation ).

Morphological/Genetic Comparisons
For both sexes, the number of inner setae on the distal segment of the third-swimming-leg exopod is known to differ within and among C. deitersi populations (Fleeger 1980Citation ) and in related species (Mielke 2000Citation ). To determine if morphological and genetic patterns correlated in C. deitersi, 51 specimens from Louisiana and 23 from Alabama were examined morphologically and genetically. The third leg was removed with a fine probe, mounted on a microscope slide, and examined with phase microscopy. The remaining copepod tissue was used for DNA extraction and genotyping by MHS-PCR. Specimens were given identification numbers upon dissection, and genetic analysis was conducted without knowledge of their geographic origins. In addition, the third-leg exopods of several specimens from California and Mexico were examined and compared with specimens from the Gulf of Mexico.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
Genotypic Variability
A total of 135 copepods (121 C. deitersi, 7 C. helobius, 2 Coullana sp., and 5 C. pacificus) were initially sequenced for one or more gene regions. The number of distinct COX-I haplotypes per locality ranged from 1 (Mazatlán) to 10 (Alabama) (table 1 ). COX-I sequences fell into one of three major types, which will be hereinafter referred to as "major types" or "major lineages." Type I was found among copepods from Alabama, and type II was found predominantly among organisms from Louisiana but also in Alabama, whereas type III was exclusively present in all copepods from California (IIIC) and Mazatlán (IIIM). A small fraction of copepods from Louisiana also harbored type I sequences.

Of all the C. deitersi of types I and II genotyped (sequencing + MHS-PCR, total N = 240), 11 (7.7%) from Louisiana harbored type I, and 132 (92.3%) harbored type II COX-I sequences. Conversely, 90 (92.8%) C. deitersi from Alabama had type I, whereas only 7 (7.2%) had type II. All COX-I sequences obtained from 15 haphazardly selected copepods genotyped via MHS-PCR (both type I and type II) corroborated the identifications obtained with the PCR-based method.

Eleven distinct 16S sequences were found among 47 C. deitersi analyzed (table 1 ). Overall, the 16S rDNA followed the same pattern as COX-I, in that sequences fell into major types analogous to those identified earlier.

We obtained nuclear rDNA sequences from 23 C. deitersi, 4 C. helobius, and 1 C. pacificus (table 1 ). The nuclear gene regions showed different levels of variation, with ITS having the highest and 5.8S having the lowest. Except for the Mexican copepods that shared two distinct ITS1 alleles but only one ITS2, the remaining copepods had the same number of ITS1 and ITS2 distinct alleles. In sharp contrast, the 5.8S rDNA showed very low variability, with only four distinct alleles among the 23 C. deitersi and two among the 4 C. helobius (table 1 ). However, no nuclear alleles were shared between the two congeneric species. In both ITS regions, alleles assorted into three extremely divergent groups, each matching the major mitochondrial types (I, II, and III) previously identified on the basis of the mtDNA sequences. On the other hand, one of the four 5.8S alleles was shared by individuals with different mtDNA types, notwithstanding the fact that in each organism the same 5.8S allele was flanked by drastically different ITS alleles falling into the divergent groups described above.

Intraspecific and Interspecific Levels of Genetic Differentiation
Levels of genetic differentiation within each major type of C. deitersi were small and typical of those found among conspecific organisms, leading to the assumption that each type represents a putative species. Mitochondrial genes were less variable (0.2%–1.7%; diagonal of table 2 ) than noncoding nuclear ITS regions (2.7%–8.5% in faster-evolving ITS1 and 0.7%–2.6% in ITS2; table 3 ). As expected, intralineage variation in the 5.8S rRNA gene was virtually absent. Although copepods from Mazatlán were unequivocally most closely related to those from California, they were quite differentiated in both mitochondrial (11% in COX-I and 5% in 16S rRNA) and nuclear (12% in ITS1 and 2.8% in ITS2) genes (table 3 ). Thus, four species may be represented within our three major lineages of C. deitersi.


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Table 2 Mean Pairwise Genetic Distances in the COX-I (above diagonal) and 16S (below diagonal) Mitochondrial Genes in Four Lineages of Cletocamptus deitersi (types I, II, IIIC, and IIIM) and in Cletocamptus helobius (CH)

 

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Table 3 Mean Uncorrected Genetic Distances (%) in the ITS1, 5.8S, and ITS2 Gene Regions in Four Lineages of Cletocamptus deitersi (types I, II, IIIC, and IIIM) and in Cletocamptus helobius (CH)

 
Further evidence that our nominal C. deitersi is composed of more than one species came from a comparison with its morphologically differentiable congener C. helobius. Organisms of the three major lineages of C. deitersi were as genetically differentiated from each other as they were from C. helobius. Corrected levels of mtDNA sequence divergence among the three major lineages were at least one, if not two, orders of magnitude higher than intralineage divergences, with values ranging from 48.7% to 88.3% (20.8%–26.4% uncorrected) in the COX-I gene and from 29.4% to 82.5% (20.3%–36.0% uncorrected) in the 16S rRNA (table 2 ). The corresponding values of indisputably interspecific (i.e., C. deitersi vs. C. helobius) differentiation were 71.9%–91.7% (23.7%–26.8% uncorrected) for the COX-I gene and 63.1%–81.4% (31.7%–35.8% uncorrected) for the 16S rRNA (table 2 ). Data from nuclear genes were consistent with mitochondrial results. For the widely divergent noncoding ITS regions, nucleotide sequences of alleles from different lineages were so different that their alignment was dubious (fig. 2 ). Conspicuous size differences (ITS1 lengths were 359–376 bp in type I, 292–300 bp in type II, 288–289 bp in type IIIC, and 284 bp in type IIIM; whereas ITS2 lengths were 221–228 bp in type I, 204–206 bp in type II, 215–219 bp in type IIIC, and 216 bp in type IIIM) and a large degree of sequence differentiation are indicative of a thorough randomization of the nucleotide sequences via mutation and recombination, such that statements of sequence homology can no longer be established confidently by alignment. Thus, no quantitative appraisal of divergence is given (table 3 ); instead, we present the imposed multiple alignment as qualitative evidence of the extent of differentiation (fig. 2 ). The alignment shows that aside from the well conserved 18S and 5.8S rDNAs, the alignability of the sequences decays rapidly in the ITS regions, but not to the same extent among the lineages of Cletocamptus, since types II and III share more structural elements. Finally, in the slowly evolving 5.8S rRNA gene, mean levels of intraspecific variation in the nominal C. deitersi were smaller (0.6%–1.5%) than those of interspecific variation (3.7%–4.6%).



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Fig. 2.—CLUSTAL W DNA multiple alignment of part of the nuclear rDNA comprising the entire internal transcribed spacers (ITS1 and ITS2) and the intervening 5.8S ribosomal RNA gene. Gene regions are indicated at the bottom of the alignment. Labels refer to GenBank accession numbers presented in table 1 . Dots represent identity with the majority-rule consensus sequence on top, and dashes represent inferred indels

 
Molecular Evolution and Phylogenetic Analyses
The 16S amplicons in C. deitersi were either 462 bp (type I) or 467–468 bp (types II and III) in length. The amplified mitochondrial COX-I fragment was 658 bp long in all organisms. Almost a third of the nucleotides were variable (228 total variable sites; the ratio of variable 1:2:3 codon positions was 43:8:177). Inferred COX-I amino acid sequences revealed that all nucleotide substitutions within types I and II were silent (details not shown). Most amino acid replacements were conservative with respect to hydrophobicity (20 out of 23; see supplementary data at http://www.molbiolevol.org). Rates of synonymous and nonsynonymous substitutions along the lineages leading to types I, II, and III were dS = 11.19, dN = 0.04 (I); dS = 1.56, dN = 0.006 (II); and dS = 1.87, dN = 0.007 (III).

Different models of nucleotide substitution were fitted to each data set. For the COX-I gene, the best-fit model was K81 (Kimura 1981Citation ) with unequal base frequencies corrected for proportion of invariable sites (I) and for rate heterogeneity among sites with a Gamma distribution (G; Yang 1993Citation ). For the 16S rRNA gene, the best-fit model was TIM (TIM+I+G; Rodriguez et al. 1990Citation ), whereas for the 5.8S rRNA gene, the best-fit model was Jukes-Cantor (Jukes and Cantor 1969Citation ) (table 4 ).


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Table 4 Parameters of the Models of Nucleotide Substitution Fit to Mitochondrial and Nuclear Genes of Cletocamptus deitersi

 
The COX-I and 16S rDNA alignments including all taxa had, respectively, 309 and 215 variable sites (treating indels as missing data), of which 275 and 180 were parsimony-informative. The strict-consensus tree of the eight COX-I most-parsimonious reconstructions obtained via branch-and-bound (length = 670 steps, consistency index [CI] = 0.72, retention index [RI] = 0.91) was completely congruent with the NJ tree (fig. 3 ). For the 16S, the NJ tree differed from the three branch-and-bound most-parsimonious trees (length = 419 steps, CI = 0.79, RI = 0.87) in that Cletocamptus was not monophyletic. Instead, C. helobius was sister to type I sequences and both were a sister group to the rest of the C. deitersi sequences (fig. 3 ). An NJ tree constrained by monophyly of C. deitersi was not significantly different from the unconstrained tree (LRT; {delta} = 2.48, P = 0.43). In the MP analyses, the monophyly of C. deitersi had a nonparametric bootstrap branch support of 64%. Trees featured extensive unresolved polytomies within each major type (I, II, and IIIC) but were otherwise completely resolved. Both mtDNA gene trees featured long branches, in excess of 30% corrected sequence divergence, separating C. deitersi types I, II, and III and the other congeneric and noncongeneric species. All bootstrap replicates recovered the monophyly of the three major mitochondrial types in both genes. Within type III sequences, the Californian haplotypes formed a reciprocally monophyletic assemblage relative to the Mexican haplotypes with very high bootstrap support. Despite overall topological congruence of the gene trees, considering that a 16S NJ tree constrained by the monophyly of C. deitersi is not significantly worse than the one shown (fig. 3 ), the two mtDNA genes were not equally powerful in resolving the relationships of the major lineages within Cletocamptus. For example, the sister taxon relationship between types II and III was very strongly supported by the NJ analysis of the 16S data set but less so by the corresponding MP analysis or by the COX-I data set (bootstrap 55%–57%). Monophyly of Cletocamptus and Harpacticoida, however, was very well supported by the mtDNA data (bootstrap > 93%).



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Fig. 3.—Neighbor-joining (NJ) phylogenetic reconstructions based on genetic distances of distinct mitochondrial cytochrome oxidase subunit I (COX-I, K81uf+I+G distances) and 16S ribosomal RNA (16S, TIM+I+G distances) haplotypes of the harpacticoid copepods Cletocamptus deitersi, Cletocamptus helobius, and Coullana sp. and the calanoid copepod Calanus pacificus. Branch lengths are proportional to genetic distance according to scales shown. Numbers on branches are nonparametric bootstrap support values (1,000 replicates) obtained with NJ (above) and with maximum parsimony (below). Localities where haplotypes were found are indicated by symbols; numbers in symbols are the numbers of copepods sharing a particular haplotype. See text for explanation of genetic distance measures. n.a. = not applicable

 
The nuclear 5.8S rDNA alignment was 166 bp long with 32 variable sites, of which only 6 were parsimony-informative. An exhaustive search produced a single most-parsimonious reconstruction (length = 33, CI = 1.0, RI = 1.0), and the NJ reconstruction was completely congruent (fig. 4 ). Unlike the mitochondrial gene trees, the nuclear 5.8S rDNA proved more powerful for supporting the monophyly of C. deitersi (fig. 4 ). Variation within C. deitersi was too small to gain any insight into the phylogenetic relationships among mitochondrial lineages.



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Fig. 4.—Neighbor-joining (NJ) phylogenetic reconstruction based on Jukes-Cantor genetic distances of distinct nuclear 5.8S ribosomal RNA alleles of the harpacticoid copepods Cletocamptus deitersi and Cletocamptus helobius and the calanoid copepod Calanus pacificus. Branch lengths represent genetic distances according to the scale shown. Numbers on branches represent nonparametric bootstrap support values (1,000 replicates) obtained with NJ (above) and with maximum parsimony (below). The corresponding mitochondrial haplotype found in the same copepods is indicated by the symbols. Numbers in symbols represent the individuals sharing the same 5.8S rRNA gene copy

 
Branch-and-bound maximum-likelihood analyses using best-fit models of substitution and one representative sequence of each Cletocamptus lineage, including C. helobius, indicated that the existence of a molecular clock could not be rejected in the mtDNA genes (COX-I: {chi}2 = 2.89, P = 0.41; 16S: {chi}2 = 6.04, P = 0.11).

Morphological/Genetic Comparisons
All specimens of C. deitersi examined in this study had indistinguishable body plans (i.e., body length and width, rostrum shape, caudal rami shape, and leg segmentation were essentially identical). Small variations in leg setation and body ornamentation have been described in species related to C. deitersi. For example, the number of inner setae on the distal segment of the third-leg exopod of Cletocamptus axi (one seta) differs from that of Cletocamptus schmidti (two setae) in both sexes (Mielke 2000Citation ). Specimens from Louisiana, California, and Mexico all had one inner seta, whereas those from Alabama had two. MHS-PCR was successful for 70 of the 74 specimens. Copepods with one inner seta on the third-leg exopod, all from Louisiana, were of mtDNA COX-I major type II, whereas those with two inner setae, all from Alabama, were of type I.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
Genetic Diagnosis of Species Boundaries
Species criteria are not necessarily mutually exclusive, and alternate criteria typically capture different aspects of the biological information present in a particular data set. We suggest that rather than use a single criterion specified a priori for diagnosing species boundaries, using multiple criteria simultaneously can produce species identifications that are more robust to the assumptions employed by different investigators. Therefore, where appropriate, we will make specific reference below to the genealogical concordance, phylogenetic, and morphological species criteria (see de Queiroz [1998Citation ] for terminology).

The nominally intraspecific genetic differentiation found in mitochondrial and nuclear gene regions among major lineages of C. deitersi represents the largest yet reported for a marine invertebrate and strongly suggests that North American lineages are composed of cryptic species. The likelihood that the large genetic differentiation is due to sequences derived from nonfunctional paralogous copies (e.g., pseudogenes), as reported for other crustaceans (Schubart, Neigel, and Felder 2000bCitation ), is negligible. This assertion is supported by patterns of nucleotide substitutions, in relation to reading frames and rates of synonymous and nonsynonymous substitutions per synonymous and nonsynonymous sites (dS/dN) (COX-I), coding versus noncoding regions (5.8S rDNA), and secondary structures (16S, 5.8S rDNA) observed in the gene regions examined (details not shown). Also, our sequence data sets are large (table 1 ), so the apparent absence of intermediate nodes in our phylogenetic reconstructions is not an artifact of inadequate sampling within our four collecting sites (e.g., Funk 1999Citation ). Instead, the three major types (I, II, and III) can be characterized as separate species using the phylogenetic-species and the genealogical-concordance criteria. In addition, our data and preliminary morphological observations (S. Gómez, personal communication) suggest that type III may be composed of two species. Admittedly, we analyzed samples from only four localities in one subcontinent of the almost cosmopolitan C. deitersi. Because our phylogenetic trees are based on a limited geographic sampling, it is possible that analysis of additional C. deitersi from other locations would uncover nodes that are intermediate in depth between those separating the major lineages and those separating individual sequences within lineages. Although such a potential result would complicate the use of sequence data for systematic purposes within Cletocamptus, it would not invalidate our major conclusions concerning the existence of deep lineages and morphological stasis.

Additional support for the assertion that our collections are composed of highly divergent species comes from comparison with orthologous sequences of a bona fide congeneric species (tables 2 and 3 ) and from comparisons with patterns of genetic differentiation found in more than 200 invertebrate taxa (table 5 ). These patterns show that levels of mtDNA differentiation among the major types in C. deitersi are comparable to those found at several supraspecific and often suprageneric levels (table 5 ). The divergence between Mexican (IIIM) and Californian (IIIC) copepods is dwarfed by that found among the major mtDNA types (I, II, and III), but it is comparable to that separating morphologically differentiated and uncontested species in other congeneric arthropods (table 5 ). In contrast, intraspecific differentiation in divergent lineages suspected of harboring cryptic or sibling species are generally considerably smaller than those found in C. deitersi (table 5 ). Levels of mtDNA divergence comparable to those in C. deitersi have been found in the freshwater amphipod Hyalella azteca and the marine harpacticoid copepod Tigriopus californicus. In H. azteca, seven mitochondrial lineages of presumed cryptic species sampled within central glaciated North America showed extreme levels of divergence (COX-I: 20.2%–27.6% corrected; Witt and Hebert 2000)Citation . In T. californicus, allopatric populations at one end of the species range in southern Baja California exhibit reproductive isolation (Ganz and Burton 1995Citation ) and very large levels of divergence (COX-I: 18%–23% uncorrected; S. Edmands, personal communication) from the rest.


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Table 5 Comparison of Genetic Divergence in Mitochondrial Genes Among Lineages of Cletocamptus deitersi and Among Other Invertebrate Taxa of Different Taxonomic Levels

 
We have not found in the literature a degree of ITS differentiation as extreme (i.e., unalignable sequences) as the one found among the putative cryptic species in C. deitersi in conspecifics and in members of closely related complexes of cryptic or sibling species. Congeneric invertebrates can sometimes show significant differentiation in the ITSs of nuclear ribosomal genes (e.g., up to 23% across taxa in Acropora; Odorico and Miller 1997Citation ). More often, however, ITS sequences show incipient levels of differentiation (<5%) among subspecific organisms or others belonging to complexes of cryptic or sibling species (e.g., black flies in the genus Simulium; Tang et al. 1996Citation ). In the ITS1 of Microarthridion littorale, the only harpacticoid ITS sequence reported to date, the largest divergence (only 10.8%) was found between organisms bearing extreme cytochrome b differentiation (ca. 30%, computed from data in Schizas et al. [1999]Citation ).

The comparisons cited above strongly suggest that genetic differentiation among the major lineages of the nominal harpacticoid C. deitersi would be reconcilable with intraspecific polymorphism only in the presence of (1) extraordinarily high rates of molecular evolution in both mitochondrial and nuclear genomes, (2) strong balancing selection, or (3) historical vicariant events that would maintain the polymorphisms. We argue that additional evidence contradicts these alternative interpretations.

First, there appears to be a strong correspondence between morphology (third-leg setation) and molecular lineage (nuclear and mitochondrial) in 70 nominal C. deitersi COX-I's examined from Louisiana and Alabama. All copepods with one inner seta on the third-leg exopod were from Louisiana and all were of type II, whereas those with two inner setae, all from Alabama, were of type I. Our molecular studies show that both type I and type II lineages are sympatric in Louisiana and Alabama, with both locations exhibiting one dominant and one rare lineage. Even though leg setation could be environmentally determined (e.g., by salinity, which is much higher at the brine stream), Fleeger (1980)Citation found that both morphotypes (one or two inner setae on the third-leg exopod) co-occur in Louisiana salt marshes. This suggests that leg setation is not a morphologically plastic nongenetic response or an adaptive character under strong habitat-specific selection that has evolved rapidly relative to the timing of cladogenetic events. This trait thus provides more evidence of concordant patterns of molecular and morphological variation, consistent with the interpretation that types I and II constitute separate species on multiple criteria. Concordance of molecular and morphological variation would not be expected if the deep phylogenetic nodes separating the major types were an artifact of high mutation rates in the gene regions examined.

Second, the suggestion that gene regions from two genomes would all be under strong balancing selection appears unlikely. Selection can maintain ancient polymorphisms within, and even between, species (Hughes and Yeager 1998Citation ). The only mechanism for maintenance of ancient alleles in mtDNA that has been reported in invertebrates is male- and female-specific mtDNA genomes associated with sex determination in some bivalves (e.g., Quesada, Wenne, and Skibinski 1999Citation ). However, this is unlikely here, since the skewed relative frequencies of type I and type II sequences between Alabama and Louisiana (i.e., type I is rare in Louisiana, but type II is rare in Alabama) suggests no connection with sex determination.

Third, it is possible that C. deitersi shows genetic signatures of historical vicariant events in the form of high levels of genetic differentiation. Such patterns have been documented for a number of coastal marine invertebrates in different regions of the world, including the harpacticoid copepod T. californicus (Burton and Lee 1994Citation ; but see Burton 1998Citation ). In these continuously distributed species, a deep genetic break coincides with a documented faunal break, reflecting the influence of historical vicariant events (phylogeographic category I of Avise [2000]Citation ). Limited geographic sampling prevents determination of whether any of our deep phylogenetic nodes correspond to known phylogeographic break points in other species. However, this scenario does not involve mechanisms preventing conspecific organisms from hybridizing in sympatry. Although present samples are limited, we did not detect heterozygosity for nuclear rDNA sequences characteristic of types I and II in either the Alabama or the Louisiana samples. This inferred lack of hybridization provides evidence against a vicariant explanation of deep phylogenetic nodes and corroborates the genealogical-concordance results. Additional nuclear genes are needed to determine the extent of reproductive isolation of sympatric types I and II, which would satisfy the biological-species criterion.

Temporal Perspective
Extensive genetic differentiation among the genetic lineages of C. deitersi suggests long times since initial divergence. Assuming that the rate of molecular evolution in the COX-I gene calibrated in other crustaceans can be applied to C. deitersi, copepods from the Salton Sea and Mazatlán appear to have shared a common ancestor between the late Miocene and the early Pliocene (table 6 ). The molecular ancestor of lineages II and III dates back to the early and middle Miocene, and the oldest divergence between lineages I and II and lineage III dates back to a time between the late Eocene and the early Miocene (table 6 ). These dates are in remarkable agreement with the fossil record. Fossilized specimens assignable to Cletocamptus have been found in sedimentary deposits dating to the middle and late Miocene (Palmer 1960Citation ). The fossils are similar to an extant species, Cletocamptus albuquerquensis (Herrick) but cannot be identified due to lack of preservation of key characters. However, close resemblance between the fossil and recent Cletocamptus suggests a morphological conservatism that has lasted for more than 10 Myr, a timescale comparable to the one suggested by the molecular data of the seemingly old cryptic species of C. deitersi. The mitochondrial 16S rDNA distances and rates yield larger estimates of divergence than the COX-I gene data (table 6 ). The discrepancy may be accounted for by a larger correction imposed by the Hasegawa, Kishino, and Yano (1985)Citation model with gamma correction for rate heterogeneity among sites (Yang 1993Citation ) on the 16S rRNA gene, particularly with respect to the rate heterogeneity among sites, and, more importantly, by the relative rates of evolution of both genes in C. deitersi, where it appears that the 16S rRNA gene evolves faster than the COX-I gene (table 2 ). This difference is likely the result of the inclusion of hypervariable regions in the molecule (i.e., loops) that are much less variable in the taxa and timescales involved in rate calibrations. Rates previously reported (cf. table 6 ) reflect that the COX-I gene evolves twice to almost six times as fast as the 16S gene. Even if the present COX-I gene sequences showed uncorrected saturation and the "true" rate of evolution was closer to the one shown by the 16S rRNA gene, this would only increase the estimates of time since divergence, making the morphological conservatism of C. deitersi even more remarkable.


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Table 6 Ranges of Divergence Since Common Ancestor Based on Corrected Sequence Divergence and Crustacean Molecular-Clock Calibrations

 
Morphological Stasis
Two types of analyses have generally been conducted to address the question of whether evolutionary processes such as drift, mutation, and selection result in a correlation between rates of morphological and molecular evolution over diverse time intervals. One approach is to collect DNA sequence or other molecular data on morphologically conservative taxa and compare the amounts of molecular divergence at particular taxonomic levels to amounts of divergence characterizing the same taxonomic levels in less conservative taxa (e.g., Wilson, Carlson, and White 1977Citation ; Avise, Nelson, and Sugita 1994Citation ; King and Hanner 1998Citation ; Jarman and Elliott 2000Citation ). These studies have provided clear evidence of a decoupling between the two rates of evolution in selected taxa. When molecular divergence times can be dated with geological evidence, the results are consistent with morphological stasis. The generality of these studies has been questioned, both on the grounds that taxa known a priori to be morphologically conservative were chosen for analysis (Omland 1997Citation ) and on the grounds that nonmorphological characters such as physiological, ecological, or behavioral traits might show changes that are correlated with molecular differences (Schubart, Neigel, and Felder 2000bCitation ). A second approach is to compare rates of morphological and molecular evolution in taxa chosen without bias regarding expectations about these rates. For example, Omland (1997)Citation found significant positive correlations between inferred morphological and molecular branch lengths in a phylogenetic study of eight taxa. Our study is somewhat different from both of these approaches in that we had no prior expectation of ancient divergence times in the presence of morphological conservatism within the nominal C. deitersi. This result may, in part, reflect developing taxonomic resolution within Cletocamptus, and retrospective analysis of our four major types (I, II, IIIC, and IIIM) has revealed subtle morphological differences among them (S. Gómez, personal communication). However, even the most genetically divergent lineages in our data set share identical body plans, and they cannot be differentiated by casual observation at magnifications of less than 100x (J.W.F., unpublished data). We conclude that the morphological stasis of these types over a time interval of at least 10 Myr is real. Given the challenge of studying evolutionary and ecological processes in these small benthic copepods, one way to increase our understanding of the mechanisms and patterns of morphological stasis from a comparative perspective would be to examine additional meiobenthic invertebrate taxa for the occurrence of cryptic species and deep evolutionary lineages.


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 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
The nucleotide alignment of the COX-I sequences submitted to GenBank is available at http://www.molbiolevol.org.


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Table 5 Continued

 


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Fig. 2 (Continued)

 

    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 
We are grateful to John McCall and Markus Wetzel for help in harpacticoid collection efforts. Debra Dexter (California) and Samuel Gómez and Ana Puello-Cruz (Mexico) made their cultured copepods available to us. Mark Ohman and Annie Townsend, at the Planktonic Invertebrates Collection of the Scripps Institution of Oceanography, provided a gift of C. pacificus specimens. Laboratory assistance from Mary Martin, Deb Taranik, and Nannette Crochet is gratefully acknowledged. Michael Hellberg, Scott Herke, Dorothy Prowell, and Mohamed Noor made useful suggestions on an earlier draft. This work was funded by Minerals Management Service contract 14-35-0001-30660. A.R.-O. wishes to dedicate his contribution to the memory of Dr. Michael M. Mullin, foremost scientist, teacher, and friend.


    Footnotes
 
Keith Crandall, Reviewing Editor

1 Abbreviations: bp, base pairs; COX-I, cytochrome oxidase subunit I; ITS, internal transcribed spacer; LSU rDNA, large-subunit ribosomal DNA; mtDNA, mitochondrial DNA. Back

2 Keywords: Cletocamptus deitersi, cytochrome oxidase subunit I 16S ribosomal DNA nuclear ribosomal DNA morphological stasis cryptic species Back

3 Address for correspondence and reprints: Axayácatl Rocha-Olivares, Louisiana State University, Department of Biological Sciences, 508 Life Sciences Building, Baton Rouge, Louisiana 70803-1715. a.rocha-olivares{at}sciencenet.com Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 literature cited
 

    Andersen, N. M., L. Cheng, J. Damgaard, F. A. H. Sperling. 2000. Mitochondrial DNA sequence variation and phylogeography of oceanic insects (Hemiptera: Gerridae: Halobates spp.). Mar. Biol. 136:421–430[ISI]

    Avise, J. C.. 2000. Phylogeography: the history and formation of speciesHarvard University Press, Cambridge, Mass

    Avise, J. C., W. S. Nelson, H. Sugita. 1994. A speciational history of "living fossils": molecular evolutionary patterns in horseshoe crabs. Evolution. 48:1986–2001[ISI]

    Baric, S., C. Sturmbauer. 1999. Ecological parallelism and cryptic species in the genus Ophiothrix derived from mitochondrial DNA sequences. Mol. Phylogenet. Evol. 11:157–162[ISI][Medline]

    Braga, E., R. Zardoya, A. Meyer, J. Yen. 1999. Mitochondrial and nuclear rRNA based copepod phylogeny with emphasis on the Euchaetidae (Calanoida). Mar. Biol. 133:79–90[ISI]

    Bucklin, A., C. C. Caudill, M. Guarnieri. 1998. Population genetics and phylogeny of planktonic copepodsPp. 303–318 in K. E. Cooksey, ed. Molecular approaches to the study of the ocean. Chapman and Hall, London

    Burton, R. S.. 1998. Intraspecific phylogeography across the Point Conception biogeographic boundary. Evolution. 52:734–745[ISI]

    Burton, R. S., B. N. Lee. 1994. Nuclear and mitochondrial gene genealogies and allozyme polymorphism across a major phylogeographic break in the copepod Tigriopus californicus.. Proc. Natl. Acad. Sci. USA. 91:5197–5201[Abstract]

    Cunningham, C. W., N. W. Blackstone, L. W. Buss. 1992. Evolution of king crabs from hermit crab ancestors. Nature (Lond.). 355:539–542[ISI][Medline]

    de Queiroz, K.. 1998. The general lineage concepts of species, species criteria, and the process of speciationPp. 57–75 in D. J. Howard and S. H. Berlocher, eds. Endless forms: species and speciation. Oxford University Press, New York

    Etter, R. J., M. A. Rex, M. C. Chase, J. M. Quattro. 1999. A genetic dimension to deep-sea biodiversity. Deep Sea Res. I Oceanogr. Res. Pap. 46:1095–1099[ISI]

    Fleeger, J. W.. 1980. Morphological variation in Cletocamptus (Copepoda: Harpacticoida), with description of a new species from Louisiana salt marshes. Trans. Am. Microsc. Soc. 99:25–31[ISI]

    Folmer, O., M. Black, W. Hoeh, R. Lutz, R. Vrijenhoek. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3:294–299[Medline]

    Foltz, D. W.. 1997. Hybridization frequency is negatively correlated with divergence time of mitochondrial DNA haplotypes in a sea star (Leptasterias spp.) species complex. Evolution. 51:283–288[ISI]

    Funk, D. J.. 1999. Molecular systematics of cytochrome oxidase I and 16S from Neochlamisus leaf beetles and the importance of sampling. Mol. Biol. Evol. 16:67–82[Abstract]

    Ganz, H. H., R. S. Burton. 1995. Genetic differentiation and reproductive incompatibility among Baja California populations of the copepod Tigriopus californicus.. Mar. Biol. 123:821–827[ISI]

    Geller, J. B., E. D. Walton, E. D. Grosholz, G. M. Ruiz. 1997. Cryptic invasions of the crab Carcinus detected by molecular phylogeography. Mol. Ecol. 6:901–906[ISI][Medline]

    Hasegawa, M., H. Kishino, T. Yano. 1985. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 21:160–174

    Heath, D. D., P. D. Rawson, T. J. Hilbish. 1995. PCR-based nuclear markers identify alien blue mussel (Mytilus spp.) genotypes on the west coast of Canada. Can. J. Fish. Aquat. Sci. 52:2621–2627[ISI]

    Hughes, A. L., M. Yeager. 1998. Natural selection at major histocompatibility complex loci of vertebrates. Annu. Rev. Genet. 32:415–435[ISI][Medline]

    Huys, R., J. M. Gee, C. G. Moore, R. Hamond. 1996. Marine and brackish water harpacticoid copepodsPart 1. Linnean Society of London, Dorchester, England

    Jarman, S. N., N. G. Elliott. 2000. DNA evidence for morphological and cryptic Cenozoic speciations in the Anaspididae, ‘living fossils' from the Triassic. J. Evol. Biol. 13:624–633[ISI]

    Jukes, T. H., C. R. Cantor. 1969. Evolution of protein moleculesPp. 21–132 in H. N. Munro, ed. Mammalian protein metabolism. Academic Press, New York

    Kimura, M.. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111–120[ISI][Medline]

    ———.1981. Estimation of evolutionary distances between homologous nucleotide sequences. Proc. Natl. Acad. Sci. USA. 78:454–458

    King, J. L., R. Hanner. 1998. Cryptic species in a "living fossil" lineage: taxonomic and phylogenetic relationships within the genus Lepidurus (Crustacea: Notostraca) in North America. Mol. Phylogenet. Evol. 10:23–36[ISI][Medline]

    Knowlton, N.. 1993. Sibling species in the sea. Annu. Rev. Ecol. Syst. 24:189–216[ISI]

    Knowlton, N., L. A. Weigt. 1998. New dates and new rates for divergence across the Isthmus of Panama. Proc. R. Soc. Lond. B Biol. Sci. 265:2257–2263[ISI]

    Knowlton, N., L. A. Weigt, L. A. Solorzano, D. K. Mills, E. Bermingham. 1993. Divergence in proteins, mitochondrial DNA, and reproductive compatibility across the Isthmus of Panama. Science. 260:1629–1632[ISI][Medline]

    Knowlton, N., E. Weil, L. A. Weigt, H. M. Guzman. 1992. Sibling species in Montastraea annularis, coral bleaching, and the coral climate record. Science. 255:330–333[ISI]

    Lee, C. E.. 1999. Rapid and repeated invasions of fresh water by the copepod Eurytemora affinis.. Evolution. 53:1423–1434[ISI]

    Mielke, W.. 2000. Two new species of Cletocamptus (Copepoda: Harpacticoida) from Galapagos, closely related to the cosmopolitan C. deitersi.. J. Crustac. Biol. 20:273–284[ISI]

    Odorico, D. M., D. J. Miller. 1997. Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution. Mol. Biol. Evol. 14:465–473[Abstract]

    Omland, K. E.. 1997. Correlated rates of molecular and morphological evolution. Evolution. 51:1381–1393[ISI]

    Palmer, A. R.. 1960. Miocene copepods from the Mojave Desert, California. J. Paleontol. 34:447–452[ISI]

    Palumbi, S. R.. 1996. Nucleic acids II: the polymerase chain reactionPp. 205–247 in D. M. Hillis, C. Moritz, and B. K. Mable, eds. Molecular systematics. Sinauer, Sunderland, Mass

    Palumbi, S. R., J. Benzie. 1991. Large mitochondrial DNA differences between morphologically similar penaeid shrimp. Mol. Mar. Biol. Biotechnol. 1:27–34[Medline]

    Patarnello, T., L. Bargelloni, V. Varotto, B. Battaglia. 1996. Krill evolution and the Antarctic Ocean currents: evidence of vicariant speciation as inferred by molecular data. Mar. Biol. 126:603–608[ISI]

    Posada, D., K. A. Crandall. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics. 14:817–818[Abstract]

    Quesada, H., R. Wenne, D. O. Skibinski. 1999. Interspecies transfer of female mitochondrial DNA is coupled with role-reversals and departure from neutrality in the mussel Mytilus trossulus.. Mol. Biol. Evol. 16:655–665[Abstract]

    Rambaut, A., N. C. Grassly. 1997. Seq-Gen: an application for the Monte Carlo simulation of DNA sequence evolution along phylogenetic trees. Comput. Appl. Biosci. 13:235–238[Abstract]

    Reid, J. W.. 1998. How "cosmopolitan" are the continental cyclopoid copepods? Comparison of the North American and Eurasian faunas, with description of Acanthocyclops parasensitivus sp.n. (Copepoda: Cyclopoida) from the U.S.A. Zool. Anz. 236:109–118

    Rocha-Olivares, A.. 1998. Multiplex haplotype-specific PCR: a new approach for species identification of the early life stages of rockfishes of the species-rich genus Sebastes Cuvier. J. Exp. Mar. Biol. Ecol. 231:279–290[ISI]

    Rodriguez, F., J. F. Oliver, A. Marin, J. R. Medina. 1990. The general stochastic model of nucleotide substitution. J. Theor. Biol. 142:485–501[ISI][Medline]

    Schizas, N. V., G. T. Street, B. C. Coull, G. T. Chandler, J. M. Quattro. 1997. An efficient DNA extraction method for small metazoans. Mol. Mar. Biol. Biotechnol. 6:381–383[ISI][Medline]

    ———.1999. Molecular population structure of the marine benthic copepod Microarthridion littorale along the southeastern and Gulf coasts of the USA. Mar. Biol. 135:399–405

    Schubart, C. D., J. A. Cuesta, R. Diesel, D. L. Felder. 2000. Molecular phylogeny, taxonomy, and evolution of nonmarine lineages within the American grapsoid crabs (Crustacea: Brachyura). Mol. Phylogenet. Evol. 15:179–190[ISI][Medline]

    Schubart, C. D., R. Diesel, S. B. Hedges. 1998. Rapid evolution to terrestrial life in Jamaican crabs. Nature. 393:363–365[ISI]

    Schubart, C. D., J. E. Neigel, D. L. Felder. 2000a.. Molecular phylogeny of mud crabs (Brachyura: Panopeidae) from the northwestern Atlantic and the role of morphological stasis and convergence. Mar. Biol. 137:11–18

    ———.2000b.. Use of the mitochondrial 16S rRNA gene for phylogenetic and population studies of CrustaceaPp. 817–830 in J. C. von Vaupel Klein and F. R. Schram, eds. The biodiversity crisis and Crustacea. A. A. Balkema, Rotterdam, The Netherlands

    Sturmbauer, C., J. S. Levinton, J. Christy. 1996. Molecular phylogeny analysis of fiddler crabs: test of the hypothesis of increasing behavioral complexity in evolution. Proc. Natl. Acad. Sci. USA. 93:10855–10857[Abstract/Free Full Text]

    Sturmbauer, C., G. B. Opadiya, H. Niederstatter, A. Riedmann, R. Dallinger. 1999. Mitochondrial DNA reveals cryptic oligochaete species differing in cadmium resistance. Mol. Biol. Evol. 16:967–974[Abstract]

    Sun, B., J. W. Fleeger. 1994. Field experiments on the colonization of meiofauna into sediment depressions. Mar. Ecol. Prog. Ser. 110:167–175[ISI]

    Swofford, D. L.. 1998. PAUP*: phylogenetic analysis using parsimony (*and other methods)Version 4.0. Sinauer, Sunderland, Mass

    Tam, Y. K., I. Kornfield, F. P. Ojeda. 1996. Divergence and zoogeography of mole crabs, Emerita spp. (Decapoda: Hippidae), in the Americas. Mar. Biol. 125:489–497

    Tang, J., L. Toe, C. Back, T. R. Unnasch. 1996. Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simuliidae) complex. Mol. Biol. Evol. 13:244–252[Abstract]

    Thompson, J. D., D. G. Higgins, T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680[Abstract]

    Wilcox, T. P., L. Hugg, J. A. Zeh, D. W. Zeh. 1997. Mitochondrial DNA sequencing reveals extreme genetic differentiation in a cryptic species complex of neotropical pseudoscorpions. Mol. Phylogenet. Evol. 7:208–216[ISI][Medline]

    Wilson, A. C., S. S. Carlson, T. J. White. 1977. Biochemical evolution. Annu. Rev. Biochem. 46:573–639[ISI][Medline]

    Witt, J. D. S., P. D. N. Hebert. 2000. Cryptic species diversity and evolution in the amphipod genus Hyalella within central glaciated North America: a molecular phylogenetic approach. Can. J. Fish. Aquat. Sci. 57:687–698[ISI]

    Yang, Z.. 1993. Maximum-likelihood estimation of phylogeny from DNA sequences when substitution rates differ over sites. Mol. Biol. Evol. 10:1396–1401[Abstract]

    ———.1997. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput. Appl. Biosci. 13:555–556

    Yang, Z., R. Nielsen. 1998. Synonymous and nonsynonymous rate variation in nuclear genes of mammals. J. Mol. Evol. 46:409–418[ISI][Medline]

Accepted for publication February 21, 2001.





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