Department of Biological Sciences, Louisiana State University
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Abstract |
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Introduction |
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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 1998
). Cryptic species are also known to occur among harpacticoid copepods (e.g., Ganz and Burton 1995
); 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. 1996
), thereby representing "pseudosibling species" sensu Knowlton (1993)
.
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)
. This species has been shown to be morphologically polymorphic within and between populations (e.g., Fleeger 1980
; Mielke 2000
). 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 1994
). 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.
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Materials and Methods |
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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)
. 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)
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 1995
). 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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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 1980
) and in related species (Mielke 2000
). 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.
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Results |
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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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Different models of nucleotide substitution were fitted to each data set. For the COX-I gene, the best-fit model was K81 (Kimura 1981
) with unequal base frequencies corrected for proportion of invariable sites (I) and for rate heterogeneity among sites with a Gamma distribution (G; Yang 1993
). For the 16S rRNA gene, the best-fit model was TIM (TIM+I+G; Rodriguez et al. 1990
), whereas for the 5.8S rRNA gene, the best-fit model was Jukes-Cantor (Jukes and Cantor 1969
) (table 4
).
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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 2000
). 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.
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Discussion |
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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 2000b
), 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 1999
). 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)
. In T. californicus, allopatric populations at one end of the species range in southern Baja California exhibit reproductive isolation (Ganz and Burton 1995
) and very large levels of divergence (COX-I: 18%23% uncorrected; S. Edmands, personal communication) from the rest.
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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)
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 1998
). 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 1999
). 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 1994
; but see Burton 1998
). 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]
). 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 1960
). 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)
model with gamma correction for rate heterogeneity among sites (Yang 1993
) 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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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Abbreviations: bp, base pairs; COX-I, cytochrome oxidase subunit I; ITS, internal transcribed spacer; LSU rDNA, large-subunit ribosomal DNA; mtDNA, mitochondrial DNA.
2 Keywords: Cletocamptus deitersi,
cytochrome oxidase subunit I
16S ribosomal DNA
nuclear ribosomal DNA
morphological stasis
cryptic species
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
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