High Cryptic Diversity across the Global Range of the
Migratory Planktonic Copepods
Pleuromamma piseki
and
P. gracilis
Kristin M. K. Halbert
1,2
, Erica Goetze
1
*, David B. Carlon
2
1 Department of Oceanography, School of Ocean and Earth Sciences and Technology, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America,
2 Department of Biology, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America
Abstract
Although holoplankton are ocean drifters and exhibit high dispersal potential, a number of studies on single species are
finding highly divergent genetic clades. These cryptic species complexes are important to discover and describe, as
identification of common marine species is fundamental to understanding ecosystem dynamics. Here we investigate the
global diversity within Pleuromamma piseki and P. gracilis, two dominant members of the migratory zooplankton
assemblage in subtropical and tropical waters worldwide. Using DNA sequence data from the mitochondrial gene
cytochrome c oxidase subunit II (mtCOII) from 522 specimens collected across the Pacific, Atlantic and Indian Oceans, we
discover twelve well-resolved genetically distinct clades in this species complex (Bayesian posterior probabilities .0.7; 6.3–
17% genetic divergence between clades). The morphologically described species P. piseki and P. gracilis did not form
monophyletic groups, rather they were distributed throughout the phylogeny and sometimes co-occurred within well-
resolved clades: this result suggests that morphological characters currently used for taxonomic identification of P. gracilis
and P. piseki may be inaccurate as indicators of species’ boundaries. Cryptic clades within the species complex ranged from
being common to rare, and from cosmopolitan to highly restricted in distribution across the global ocean. These novel
lineages appear to be ecologically divergent, with distinct biogeographic distributions across varied pelagic habitats. We
hypothesize that these mtDNA lineages are distinct species and suggest that resolving their systematic status is important,
given the ecological significance of the genus Pleuromamma in subtropical-tropical waters worldwide.
Citation: Halbert KMK, Goetze E, Carlon DB (2013) High Cryptic Diversity across the Global Range of the Migratory Planktonic Copepods Pleuromamma piseki and
P. gracilis. PLoS ONE 8(10): e77011. doi:10.1371/journal.pone.0077011
Editor: Ahmed Moustafa, American University in Cairo, Egypt
Received June 20, 2013; Accepted August 28, 2013; Published October 22, 2013
Copyright: ß 2013 Halbert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Science Foundation grant OCE-1029478 and a Waitt Foundation – National Geographic grant (W119-10) to E.
Goetze. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: egoetze@hawaii.edu
Introduction
Plankton are by definition weak swimmers, and their horizontal
movement in the ocean is largely controlled by ocean circulation
and current structure. Not surprisingly, holoplanktonic organisms
therefore are often expected to experience high gene flow among
populations, and exhibit little to no genetic differentiation across
their distributional range [1–3]. However, recent molecular studies
on holoplankton are finding that many nominal species are
composed of a number of genetically divergent clades with highly
similar morphology, with examples now reported from nearly all
pelagic metazoan phyla as well as a number of eukaryotic
phytoplankton and protistan groups (e.g., chaetognaths, [4,5];
copepods, [6–8]; pteropod molluscs, [9,10]; cnidarians, [11,12];
coccolithophores, [13]; diatoms, [14]; foraminifera, [15–17]). In
some cases, these studies are finding that species initially described
to be cosmopolitan in distribution, with ranges spanning multiple
ocean basins, in fact consist of a mosaic of evolutionarily divergent
populations that are restricted in distribution to particular pelagic
habitats. For example, the common planktonic copepod Rhinca-
lanus nasutus was once thought to be cosmopolitan in broadly
eutrophic waters worldwide [18–20], but is now well-understood
to consist of a complex of species, with many lineages occurring in
only a single coastal upwelling ecosystem [6,21]. Such observations
challenge the common perception of zooplankton as highly
dispersing species that are genetically connected throughout
widespread oceanic distributions [22], and emphasize that
regional, rather than basin-scale, oceanographic processes may
be the primary drivers for these populations.
Although relatively few prior studies have fully characterized
zooplankton cryptic species complexes, results to date suggest that
cryptic species can be recently or deeply divergent, occur in
allopatry, parapatry or sympatry with close congeners, and there
can be many or few cryptic species within a given species complex
[6,7,12,21,23–30]. Cryptic species also can be found within species
that are common in the ocean’s surface waters (e.g., this study;
[23]), as well as in species that are relatively rare [28]. Because
cryptic species can heavily bias our perceptions of large-scale
biogeographic patterns [31,32], it is important to characterize,
where possible, the true diversity within common and ecologically
important groups. Accurate knowledge of zooplankton species
diversity is also fundamental to mechanistic understanding of
pelagic food web structure and function, one of the primary goals
of biological oceanography.
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Genetic data have been central to the discovery of evolutionarily
divergent lineages within zooplankton species, in particular given
high morphological stasis in many copepod groups [33–36].
Because many of the newly discovered zooplankton lineages are
very divergent genetically, species boundaries have been effectively
detected using relatively few molecular markers [6,23,24]. Multi-
locus genetic data are particularly important in cases where
genetic differentiation among species is comparable to that among
populations, and it is difficult to detect the transition to non-
reticulating gene trees that occurs at the separation into distinct
species. There also are some reported cases of deeply divergent
mtDNA lineages within single species, and multi-locus data are
valuable for detecting these patterns (e.g., [37,38]). A number of
studies of marine zooplankton using multiple genetic markers have
found congruence and support for the inference of distinct cryptic
species across different genome regions [6,23,30], while other
studies have found broadly concordant patterns but lower
resolution at nuclear markers, even in cases where observed
divergence at mitochondrial markers was quite high [30,39,40]. In
sum, although single marker datasets have significant limitations
(e.g., [41]), the observation has been that mitochondrial DNA
sequence markers were informative in other genetic studies of
zooplankton cryptic species complexes, in part due to the fact that
in these under-studied pelagic invertebrate groups, many very
divergent lineages remain to be discovered and described.
Here we investigate global diversity within the nominal species
Pleuromamma piseki and P. gracilis, two dominant members of the
migratory zooplankton assemblage in subtropical and tropical
waters worldwide. These species co-occur and are abundant in a
range of pelagic ecosystems, though they have sometimes been
reported to have slightly distinct oceanographic distributions (e.g.,
[42–44]. Recent work reports P. piseki and P. gracilis to occur in
oceanic subtropical waters (central gyres), but P. gracilis also often
occurs in high abundance in subtropical convergence and
transitions zones (e.g., Gulf Stream extension; [43]). In the
subtropical North Pacific, 76% of the animals collected in the
euphotic zone at night are members of the genus Pleuromamma,
with P. piseki and P. gracilis dominating the 0.5–1 mm size fraction
[45,46]. Both species are diel vertical migrators (DVM) and they
have similar depth distributions, with adults in the 0–100 m layer
during the nighttime and at 200 to .400 m depths during the
daytime [44,47–50]. As a result of this migratory behavior, these
species play an important role in marine biogeochemical cycles
(e.g., [51–53]), and actively export significant fractions of carbon
and nitrogen from surface layers into the upper mesopelagic zone
[45]. Both species are omnivores [54], and they feed only at night
while in near-surface layers [48,50].
The systematics of the genus Pleuromamma is considered well-
resolved (11 species; [42,55]), and only a single new species has
been described since 1950, P. johnsoni [56]. In the most recent
comprehensive revision of the genus, Steuer [42] described three
forms within P. gracilis s. l.; forma minima, forma Piseki, and forma
maxima. Forma Piseki was elevated to species status by Farran
[57], while the names f. minima and f. maxima are not in
contemporary use (e.g., [55]; f. minima considered synonymous
with P. gracilis s. s.). Forma minima was initially described as being
circumglobal in subtropical and tropical waters (Karte 14 in Steuer
1932), and f. maxima occurred predominantly, though not
exclusively, in the southern transition zone of the Atlantic, Pacific
and Indian Oceans. Morphological differentiation between P.
piseki, P. gracilis, and the only other described small-bodied species
in the genus, P. borealis Dahl 1893, is relatively slight [55,58,59],
and males and juveniles of these species are often lumped in
ecological studies due to difficulties in identification [44,49]. One
prior study of intraspecific morphological variation within P. piseki
and P. gracilis did not find evidence of additional undescribed
species ([60], N = 17, 18 individuals), but the only prior study to
report genetic data for these species did find unusually high genetic
divergence within P. piseki (N = 2 specimens; [61]).
Our initial goal was to assess the population genetic structure of
Pleuromamma piseki across its global distribution in tropical and sub-
tropical waters, in order to better understand gene flow and
population connectivity in marine holoplankton. However, our
initial phylogenetic results based on mitochondrial cytochrome c
oxidase subunit II (mtCOII) DNA sequences revealed divergent
genetic clades within this nominal species. Our next objective then
was to characterize the diversity within this newly discovered
cryptic species complex, and determine the phylogenetic relation-
ship of the described species P. piseki and P. gracilis to the
undescribed genetic lineages present in the group. We also sought
to characterize, to the extent possible, the oceanographic
distribution of all lineages in the species complex. We conducted
preliminary efforts to detect morphological differentiation between
genetic lineages, in order to find potential characters for species
identification. And finally, due to problems with polymerase chain
reaction (PCR) amplification of orthologous mtDNA gene copies,
the final goal was to verify that the genes being amplified were
functional mitochondrial copies of mtCOII. Here we report on the
global diversity and distribution of genetic lineages within the P.
piseki – P. gracilis species complex, based on 522 specimens
collected throughout the Atlantic, Pacific, and Indian Oceans.
Materials and Methods
Sample Collection and Specimen Identification
Zooplankton were collected in bulk plankton samples from 32
locations in the Atlantic, Pacific, and Indian Oceans (Table 1,
Fig. 1). Permits were not required for these collections, and the
work did not involve endangered or protected species. Sampling
depths varied across cruises. Oblique tows were made on the
ACE-ASIA and STAR00 cruises sampling between 200 m and the
surface. Plankton tows on most other cruises sampled down to
.700 m (Table 1). The majority of these samples were obtained
by oblique tows of either a 0.71-m diameter bongo net or a 1-m
ring net (202–333
mm mesh). One sample from the North Pacific
(S230-037-TT) was collected in an oblique tow with a Tucker
trawl (333
mm mesh), to a maximum depth of 530 m. Bulk
plankton were preserved in 95% non-denatured ethyl alcohol,
changed to new alcohol within 24 h of collection, and maintained
at 220uC to minimize degradation of DNA. Specimens were
sorted from bulk plankton samples in the laboratory. Only adult
females were included due to the relative rarity of males in our
samples, and the reported difficulty in distinguishing males of the
small-bodied species in this genus.
This study initially focused on Pleuromamma piseki. This species is
characterized by relatively small body size (1.7–2 mm adult
females), a pigment spot on the right lateral prosome, adult
females that have a broad black pigmented area around the
copulatory pore, a marked groove on the left side of the genital
double-somite close to the posterior margin, and the anal somite is
parallel-sided in dorsal view [55,57,62]. However, after our initial
genetic results indicated a number of genetically divergent clades
within P. piseki, we broadened the taxonomic coverage of the study
to include Pleuromamma gracilis. Specimens of P. gracilis (N = 23)
from three regions in the Indian (VANC10MV-09), North Atlantic
(AMT20-16, 17, 18), and North Pacific Ocean (S226-12-MN,
Table 1) were included that appeared to have classic morpholog-
ical traits for P. gracilis (‘gracilis-like’). The black, pigmented area
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Table 1. Summary of 522 specimens collected from 32 locations included in this study of the P. piseki – P. gracilis species complex.
Cruise Station
Ocean Depth
Lat Long
Collection Clade Counts
Region (m) Date Total A1 A2 B1 B2 C D E F G H I J K L
0106 Tran CAN 1 NA 400 29.37 213.48 6/22/01 22 14 8
MP3 12-06-00 NA 800 29.57 245.03 7/2/01 19 13 6
MP3 14-01-00 NA 700 12.04 255.26 7/8/01 23 11 8 1 1 2
AMT20 16 NA 300 13.45 238.95 10/30/10 1 1
AMT20 17 NA 300 10.57 232.00 10/31/10 6 6
AMT20 18 NA 300 7.81 230.16 11/1/10 2 2
AMT20 32 SA 200 241.66 245.09 11/20/10 10 10
AMT20 33 SA 200 244.20 248.94 11/21/10 7 2 5
Ace-Asia 8 NP 200 31.24 173.92 3/22/01 16 8 3 1 2 2
Ace-Asia 14 NP 200 32.86 149.52 3/29/01 24 14 8 2
S226 12-MN NP 450 10.78 2161.22 12/3/09 6 1 3 2
S226 21-MN NP 316 5.02 2161.93 12/9/09 19 1 8 10
S226 48-MN NP 287 13.47 2155.32 12/26/09 21 19 2
S230 037-TT NP 750 38.12 2155.26 7/10/10 19 14 5
HOT201 ZP890 NP 160 22.75 2158.00 5/29/08 9 9
STAR00 M00-49 NP 200 12.49 2141.59 9/4/00 21 6 6 1 7 1
STAR00 M00-65 SP 200 24.44 2124.33 9/12/00 21 2 2 1 4 10 2
COOK14MV 5 SP 950 223.52 2177.05 10/7/01 20 1 14 4 1
COOK14MV 23 & 22 SP 950 231.59 2177.23 10/17/01 23 4 18 1
COOK14MV 33 SP 950 219.19 2172.54 10/24/01 24 3 19 1 1
DRFT07RR 5 SP 910 231.21 2127.50 12/20/01 23 3 19 1
DRFT07RR 8 SP 950 234.02 2140.03 12/23/01 21 5 14 2
DRFT07RR 18 SP 1040 240.53 2173.00 12/28/01 6 6
VANC10MV 2 IO 920 235.07 24.50 5/16/03 22 9 6 2 4 1
VANC10MV 6 IO 910 234.05 40.51 5/20/03 24 13 7 1 2 1
VANC10MV 9 IO 950 231.83 52.61 5/23/03 26 12 5 5 3 1
VANC10MV 11 IO 950 229.85 59.84 5/25/03 24 18 4 2
VANC10MV 16 IO 950 2
19.75 78.01 5/30/03 6 5 1
VANC10MV 20 IO 950 213.96 89.94 6/5/03 24 4 2 1 17
VANC10MV 23 IO 850 212.22 96.79 6/7/03 17 1 3 7 6
VANC10MV 25 IO 1070 213.85 109.04 6/10/03 16 16
Total 522 160 44 58 12 89 2 24 42 69 2 6 9 4 1
The numbers of specimens (N) from each station are categorized by clade identification, as shown in figure 2. NA = North Atlantic, SA = South Atlantic, NP = North Pacific, SP = South Pacific, IO = Indian Ocean. Depth is maximum
depth of tow (m).
doi:10.1371/journal.pone.0077011.t001
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around the copulatory pore was small in these specimens, in
comparison to P. piseki, and located near the distal end of the
genital boss. Pleuromamma gracilis also lacks the marked groove on
the left side of the genital double-somite that is parallel and close to
the posterior margin and body size is slightly smaller in P. gracilis
than in P. piseki. Although common in our material, P. borealis was
not included, given our initial focus on P. piseki. In expanding the
taxonomic coverage of the study, we noticed that morphological
variation among small-bodied Pleuromamma specimens was higher
than described in the prior literature. Therefore, we made
observations on morphological characters in 199 specimens prior
to DNA extraction, including measurements of body size,
variation in the shape of the genital double-somite in lateral view,
the shape and location of the black genital pigment spot, and the
presence of a groove on the left side of the genital somite (not all
characters were recorded for all specimens). The prosome length
(PL) was measured for all specimens in right lateral view, and the
total length (TL) was measured on straight-bodied individuals. We
also recorded the location of the pigment spot on the prosome
(right/left), in order to assess for any variation in this trait
(previously reported at low frequency in P. piseki, [42,63]). Digital
images and paired mtCOII DNA sequence data were obtained
from 18 specimens collected at three locations (DRFT07RR-18,
AMT20-33, VANC10MV-16), for preliminary examination of
morphological traits that differentiate genetic clades. Images of
specimens were also taken from two additional sampling sites,
S226-48MN and COOK14MV-33, but these specimens have no
paired DNA sequence data. Clade assignment for these specimens
is based on available mtCOII data from other specimens from the
same location (clades A and C, clades defined in Fig. 2). Digital
images of the posterior prosome and urosome were taken with a
Leica MZ9.5 stereomicroscope and SPOT Insight Mozaic
camera, with animals oriented in dorsal, ventral and right lateral
views. Morphological observations reported here are preliminary,
and are included in order to indicate characters that may prove
informative in future systematic studies of this group. We tested for
median differences in prosome length (PL) between genetic clades
using a Kruskal-Wallis one-way analysis of variance (ANOVA; by
ranks) followed by multiple comparison procedures (Dunn’s
methods). A total of eight clades with six or more measured
specimens each were included in this analysis (clades A, B, C, E, F,
G, I, J). Clades A1 and A2, and B1 and B2 were combined.
DNA Extraction and PCR Amplification
Genomic DNA (gDNA) was isolated from individual adult
female copepods using the DNeasy Blood and Tissue kit (Qiagen).
The manufacturer’s protocol was followed, with the following
modifications: the 55uC lysis incubation step was decreased to
1 hr, the ATL buffer and proteinase K volumes were reduced by
half, to 90
ml and 10 ml respectively, and the elution incubation
step was lengthened to 10 min.
The mitochondrial gene cytochrome oxidase subunit II
(mtCOII) was chosen as the primary genetic marker for this
study. For initial mtCOII primer design, an alignment was created
that included mtCOII data from E. bungii [64] as well as
Pareucalanus attenuatus, Euchaeta rimana, and Haloptilus longicornis
(Halbert & Goetze unpub). One set of universal primers, COIIF3
[59– TTT GGT CTA CAG GAT GCT AAC TC –39] and
COIIR3 [59– GGG ACT ATA TAG GCA TCA AAC TC –39],
were developed that amplified a 326-bp fragment of mtCOII.
Using this initial sequence, a species-specific primer, PLPICOIIFC
[59– ATT AAC TAT TAA AGC GCT TGG –39], was designed
to amplify the remainder of the mtCOII gene (401 bp), 159-bp of
a non-coding intergenic region, adenosine triphosphate synthase
subunit 8 (ATP8, 164-bp), and 169-bp of adenosine triphosphate
synthase subunit 6 (ATP6). Using these data, the species-specific
primer PLPIATP8R1 [59 – ACC ACA AGC CAG TTT ATT
GGT GA –39] was designed and used with PLPICOIIFC for
routine amplification of a 558–583-bp mitochondrial fragment
consisting of the following genes (from 5-39): 401-bp of COII, the
intergenic region, and 12-bp of ATP8. We obtained sequence data
for this region from a total of 522 specimens. This primer set
amplified a single-sized product in the majority of specimens,
however for , 7% of specimens, double-banded products where
visualized on agarose gels (not sequenced). In order to determine
clade membership of these specimens, primers PLPICOIIFC and
COIIR10 (59 – TCC TCA TGT ATA CAA ATT ACT CAA –39)
were used to amplify mtCOII in eight animals (376-bp).
Figure 1. Distribution of sampling locations (red circles) and the frequency of individuals from different genetic clades in the
Atlantic, Pacific, and Indian Oceans (pie charts), based on gDNA amplifications of mtCOII. Total sample sizes (n) for each ocean region are
given next to the pie charts. Clade colors are defined in figure 2. The cruise and station number for sample sites are listed next to the symbols (detail
in Table 1).
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PCR amplification was performed in 20 ml reaction volumes
with Bioline BIOLASE DNA polymerase (0.24
ml, initial concen-
tration 5 U/
ml), NH
4
-based Reaction Buffer (2 X), MgCl
2
(2 mM),
dNTPs (0.2 mM), forward and reverse primer (0.3
mM each), and
DNA template (,200 ng, 2.4
ml). Amplification cycles included a
DNA denaturation step at 94uC for 2 min, followed by 35 cycles of
30 s denaturing at 94uC, 30 s annealing at 55uC, 1 min of
extension at 72uC, with a final extension at 72uC for 4 min. PCR
products were purified using shrimp alkaline phosphatase and
exonuclease I (USB Corporation) for 30 min at 37uC. Sequencing
reactions were performed using BigDye terminator chemistry, and
were analyzed on an ABI 3730XL capillary-based sequencer. Both
strands were sequenced for all specimens.
Verifying Functional Gene Copies
Our initial genetic results indicated a number of highly
divergent lineages within Pleuromamma piseki. Because NUMTs
are common in crustaceans [65–67], we tested the possibility that
we were including sequence from nuclear pseudogenes. To test for
NUMTs, we sequenced PCR products from both genomic DNA
Figure 2. Bayesian phylogeny of 259 unique mtCOII haplotypes in the
P. piseki- P. gracilis
species complex (610-bp). For clarity, five
major clades are condensed into colored triangles on the tree (color as in Fig. 1). These 5 clades are shown in detail in figures 3 & 4. Bayesian posterior
probability values are given above the branches, and bootstrap values from maximum likelihood (ML) analyses are given below the branches, when
that node was supported in both Bayesian and ML analyses. Bold text indicates haplotypes that were sampled more than once. The tree is rooted,
with Pareucalanus attenuatus defined as the outgroup (labeled PAAT; Calanoida, Eucalanidae). The scale bar is in units of substitutions per site (0.7).
Shape symbols beside the haplotype identifiers indicate morphological traits of one or more specimens of that haplotype: triangle = P. piseki – like
characters, circle = P. gracilis – like characters, square = ‘clade G – like’ characters (see Results for more detail). Morphological trait observations for
major clades A, B, C, F, G are shown in figures 3 & 4.
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and mRNA transcripts of mtCOII (RT-PCR). Both gDNA and
RNA were isolated from 17 individual copepods from three
mtDNA clades that were collected in the subtropical North Pacific
(station Aloha, 22.75uN 158uW, 11/26/11), preserved in RNA-
later (Sigma-Aldrich), and stored at 280uC. DNA and RNA were
separated in different aqueous phases through the addition of
Triazol (Sigma-Aldrich) and chloroform. DNA was precipitated
with 95% EtOH, washed twice with 0.1 M sodium citrate - 10%
EtOH and twice with 75% EtOH, and resuspended in 8 mM
NaOH, 0.1 M HEPES. Total RNA was precipitated with
isopropanol, washed with 75% DEPC-EtOH, resuspended with
sodium citrate (1 mM, 6.4 pH, AMBION), and treated with
DNase (TURBO DNA-free, AMBION). Reverse Transcriptase
PCR (RT-PCR) was performed to synthesize cDNA from RNA
using SUPERSCRIPT III (Invitrogen) following the manufactur-
er’s recommended protocol. We used the primer PLPICOIIRC
[59 – TTA AAA AAT CAT TTG TCC TTA CCA C –39] for
cDNA synthesis. The reaction was incubated at 55uC for 50 min,
followed by 70uC for 15 min. Complementary DNA synthesized
from RNA extractions were PCR amplified for mtCOII using
primers PLPICOIIFE [59 – CCC TAT TAT GGA AGA GCT
AA –39] and PLPICOIIRC, both of which are located within the
coding region of mtCOII. Genomic DNA extractions were
amplified using primers PLPICOIIFC and PLPIATP8R1 for the
entire mtCOII – ATP8 region, including both coding and non-
coding regions; this is the same fragment used for gDNA
amplifications in all other specimens. PCR also was conducted
on extracted RNA in order to verify the absence of contaminating
gDNA in these samples. The same PCR cycling conditions were
used as described above. Finally, we also chose three individuals in
which we clearly amplified different sized fragments, and cloned
and sequenced the PCR products in order to characterize
potential NUMTs (see Text S1).
Sequence Data Analyses and Phylogenetics
Sequences were edited using Geneious (v.5.1.6, Biomatters), and
base calls were confirmed by aligning both strands. To assess
phylogenetic relationships among specimens, maximum likelihood
(ML) and Bayesian trees were inferred from 259 unique
haplotypes that appeared to be functional gene copies based on
the absence of premature stop codons in the amino acid
translation. ML analyses were conducted using RAxML v.7.2.8
[68] and the general time reversible (GTR) model of nucleotide
substitutions with the invariant (I) model of rate heterogeneity was
selected as the best-fit model for the data (using MEGA version 5;
[69]). The data set was partitioned into two regions: mtCOII (396-
bp) and the intergenic spacer +7-bp of ATP8 (152 to 177 bp), in
order to allow substitution rates to be estimated and optimized for
each partition individually. Nodal support was assessed by
bootstrapping across nucleotide sites with 1000 replicates.
Bayesian trees were inferred with MrBayes v.3.2.0 [70,71]. We
selected substitution models for the different partitions using the
Akaike information criterion corrected for finite sample sizes
(AICc) as implemented in MEGA5 [69]. The best fitting model was
found to be GTR with gamma (G) distributed rates for the first
partition and GTR+G+I for the second partition. Four indepen-
dent chains were run with a heating parameter of 0.2, and 25% of
trees were discarded for the burn in. Clades with bootstrap support
or Bayesian posterior probabilities of .70% were considered well
supported. A second Bayesian tree was inferred that included
cDNA sequences that are known to be functional. The purpose of
this tree was to verify clades that are real taxonomic entities, by
comparing the placement of cDNA and gDNA sequences. All trees
were rooted with Pareucalanus attenuatus (Calanoida, Eucalanidae).
To quantify genetic distances between clades, the evolutionary
divergence over sequence pairs between and within clades were
averaged using the Kimura 2-parameter model (in MEGA5).
Evolutionary divergence values are reported as the percentage of
base substitutions per site, averaged over all sequences pairs
within/between clades.
Geographic Distribution and Structure
Global distribution patterns of P. piseki – P. gracilis genetic clades
were examined in a number of ways. First, we mapped the
distribution of all clades in the global ocean by plotting the
presence/absence of each genetic clade for each collection site
(using m_map, Matlab v. 7.14). These plots give initial insights into
the biogeographic distribution of these genetic populations, but are
preliminary, particularly in ocean regions where clades were
absent and sampling coverage was low. Second, a contingency
analysis was conducted to statistically test for non-random spatial
structure in the frequency of genetic clades across all oceans (using
R v.2.10.1). Clades A, B, C, F, and G were included, and
geographic regions were categorized as Indian, North Pacific,
South Pacific, North Atlantic, and South Atlantic Ocean.
Finally, we examined the population structure within clade A
across the Indian, Pacific, and Atlantic Oceans, given the
cosmopolitan distribution and higher sample size for this clade
(total N = 204). We used the estimator h
ST
to make pairwise
comparisons among our 13 localities and an analysis of molecular
variation (AMOVA, [72]) to quantify the amount of genetic
variation partitioned between different levels of hierarchical
subdivision. Populations were grouped by ocean basin: Indian
Ocean - four stations, North Pacific Ocean – six stations, North
Atlantic Ocean – three stations. The number of permutations used
for hypothesis testing was 20,000. Analyses were conducted using
Arlequin v3.5.1.3 [73]. Q-values [74] were determined for all P-
values ,0.05 to determine the false discovery rate (FDR) in tables
of pairwise comparisons. To calculate Q-values, we used the
software Q-VALUE written in R and available from John Storey
(http://genomics.princeton.edu/storeylab/qvalue/).
Results
Sequence Data and Descriptive Statistics
A total of 522 specimens from 32 locations were successfully
sequenced for the targeted mtDNA region using primers
PLPICOIIFC and PLPIATP8R (Table 1). The fragment length
ranged between 558 and 583 bp, with an average of 570 bp, due
to the varying length of the intergenic region. In total, we sampled
268 unique haplotypes with 214 singletons and 54 haplotypes
occurring more than once in the global data set. The number of
haplotypes occurring in a single location ranged from 2 to 19, with
an average of 12.5 haplotypes. An alignment that included 399-bp
of mtCOII, the intergenic region, and 7 bp of mtATP8 was used
in phylogenetic analyses (putative functional copies). Within the
final alignment, there were 262 variable sites; 52 of these were
single substitutions and 207 were parsimony-informative. The
number of synonymous and non-synonymous nucleotide substitu-
tions was 380 and 75, respectively. We also generated an
alignment including cDNA sequences, and the final length of this
alignment was 612-bp (data are available under GenBank
accession numbers KF006539– KF006807).
Phylogenetic Trees – Deep Mitochondrial Clades
Twelve genetically divergent mitochondrial clades were found
within the Pleuromamma piseki – P. gracilis species complex (Fig. 2).
All 12 of these clades were well supported in Bayesian
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phylogenetic analyses (posterior probabilities .0.70) and in most
cases also by maximum likelihood methods, indicating reciprocal
monophyly for nearly all of these clades (Figs. 2, 3, 4). Of these 12,
the five dominant clades, A, B, C, F and G, contained the majority
of the genetic diversity sampled in this study, as well as the
majority of specimens (Fig. 2, colored clades; Figs. 3, 4).
Evolutionary divergence between these five dominant clades
ranged from 6.3% (clades A & F; K2P-corrected distance) to
Figure 3. Bayesian mtCOII phylogeny of
P. piseki – P. gracilis
clade A, with sub-groups A1 and A2 as shown. Bayesian posterior
probability values are given above the branches, and bootstrap values from ML analysis are given below the branches, when that node was
supported in both Bayesian and ML analyses. Bold text indicates haplotypes that were sampled more than once. The scale bar units are in
substitutions per site. Sample identifiers begin with the ocean basin that individuals were sampled from, IO = Indian Ocean, NP = North Pacific,
SP = South Pacific, NA = North Atlantic, SA = South Atlantic. Shape symbols indicate morphological traits, as defined in the legend.
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16.99% (clades C & G), with an average divergence of 11.44%
between clades (Table 2). Clades C, E and L, in particular, were
highly divergent from all other clades (.12%, Table 2). Within-
clade divergences were an order of magnitude lower on average,
and ranged between 0.24% (clade J) and 4.4% (clade B), with an
average of 1.52% between haplotypes within the same clade
(Table 2). The remaining seven clades, K, I, H, D, E, L and J, also
were highly divergent from their closest relatives, for example
clades K and D were placed within the basal polytomy for the
species complex and were 6.75–17.11% divergent from all other
groups (Fig. 2, Table 2). However, these clades were rare in our
material, and we sampled only 1–13 individuals from each of these
clades (1–5 haplotypes). For example, the unique sequence that
constitutes clade L was sampled in only a single animal (Indian
Ocean), but was 15.7% divergent from clade C, its closest relative
(Table 2). Clade K also was represented by a single haplotype that
was sampled in 4 animals collected in the subtropical North Pacific
(2 locations, Table 1).
Major clades A and B both contained well-supported sub-
groups (Figs. 3, 4A). In clade A, four sub-groups were well
supported with Bayesian posterior probability values of .0.80
(Fig. 3A). The largest subgroup within clade A, referred to as A1
(Fig. 3), was genetically more divergent from the other sub-groups
in clade A (2.5%–3.3%) than were the other three sub-groups
(0.85%–1.5%). Therefore, these latter three groups were consid-
ered together as sub-group A2 (Fig. 3). The evolutionary
divergence between sub-groups A1 and A2 was 3.04% (Table 2).
Clades A1 and A2 were composed of 160 and 44 specimens,
respectively. Clade B was composed of two well-supported sub-
groups (Fig. 4A). Group B1 had high phylogenetic support (1.0
posterior probability, Fig. 4), while sub-group B2 was not as well
supported due to the outlier sample IO-118 (0.65 posterior
probability, Fig. 4). The posterior probability of this sub-group
increased to 0.97 when this haplotype was excluded. Sub-group B2
had the highest within-group divergence in comparison to all other
clades and sub-groups (4.2%; Table 2); even if the outlier sequence
IO-118 was excluded, the within-group divergence was still the
highest at 3.8%. Divergence between B1 and B2 was 6.46%
(Table 2). The haplotypes in clades B1 and B2 were sampled in 58
and 10 specimens, respectively.
The topology of the Bayesian mtCOII tree for the whole species
complex was a basal polytomy, with six groups ranging from low
to modest support (Fig. 2). One well supported branch contained
clades I, G, and H (0.98 posterior probability), with animals
sampled in the tropical and eastern Pacific as well as the Indian
Ocean (Table 1). A second group with modest support included
clades E, C, L, and B (0.74 posterior probability); Bayesian
analyses provided some support for placement of clade J within
this group (0.63 posterior probability), but this node was not
supported in ML analyses.
Morphological Characters and Species Boundaries
The described species P. piseki and P. gracilis did not form
monophyletic groups, rather, specimens with morphological traits
characteristic of each described species were distributed through-
out the phylogeny, sometimes co-occurring within well-resolved
clades. Specimens identified as P. piseki, based primarily on the
shape and size of the genital pigment spot and presence of the
groove on the left side of the genital double-somite (ventral view,
diagnostic trait), were placed with high support in clades A, B, C,
F, and J (Figs. 2, 3, 4). Although clades A, C and J contained only
animals identified as P. piseki, clades B and F each contained a
Figure 4. Bayesian mtCOII phylogenies of four clades in the
P. piseki
–
P. gracilis
species complex. (A) P. piseki – P. gracilis clade B, with
sub-groups B1 and B2 as shown, (B) P. piseki – P. gracilis clade F, (C) P. piseki – P. gracilis clade C, and (D) P. piseki – P. gracilis clade G. Bayesian
posterior probability values are given above the branches, and bootstrap values from ML analysis are given below the branches, when that node was
supported in both Bayesian and ML analyses. Bold text indicates haplotypes that were sampled more than once. The scale bar units are in
substitutions per site. Sample identifiers begin with the ocean basin that individuals were sampled from, IO = Indian Ocean, NP = North Pacific,
SP = South Pacific, NA = North Atlantic, SA = South Atlantic. Shape symbols indicate morphological traits, as defined in the legend.
doi:10.1371/journal.pone.0077011.g004
Table 2. Evolutionary divergence between major clades of the P. piseki–P. gracilis species complex.
A1 A2 B1 B2 C D E F G H I J K L
A1 0.97
A2 3.0 1.29
B1 9.2 8.1 2.59
B2 9.3 8.5 6.5 4.2
C 15.2 15.5 14.2 14.4 1.19
D 8.7 7.6 10.5 9.5 15.8 2.9
E 13.3 13.3 12.9 12.4 15.8 14.3 0.49
F 6.6 5.8 9.5 9.3 15.4 8.3 13.5 2.28
G 8.4 8.1 10.3 11.0 17.0 10.4 15.0 9.2 0.82
H 6.6 5.4 9.4 10.1 16.2 8.1 14.3 6.5 6.1 0.36
I 7.0 5.5 9.2 9.6 16.7 8.6 14.7 7.0 8.0 5.9 0.89
J 8.1 7.9 9.1 9.0 15.9 8.3 12.8 7.1 10.4 9.1 8.6 0.24
K 7.9 6.8 10.4 10.2 17.1 10.0 14.8 8.1 10.4 7.7 8.6 9.8 N/A
L 14.5 14.9 13.3 13.4 15.7 16.3 16.3 14.7 16.1 15.3 16.4 15.7 16.6 N/A
Numbers along the diagonal represent the within-clade divergence for each clade. The analysis included 260 unique mtCOII ha plotypes. Clades A–L are defined in
figure 2. Values are given in percentages. N/A = not applicable (single haplotype found).
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mixture of specimens with morphological characters typical for P.
piseki and P. gracilis (Figs. 2, 3, 4). Pleuromamma gracilis animals were
placed in clades E, B, F, and G, with clade E and sub-group B2 the
only clades that had exclusively P. gracilis-like animals. These
results suggest that the morphological characters on the genital
double-somite that are currently used to identify P. piseki and P.
gracilis are incongruent with the mtDNA data.
Our preliminary morphological observations suggest that there
may be traits that distinguish the genetic clades. Clade E had
significantly larger median prosome length in comparison to all
other clades, with the exception of Clade G (Kruskal-Wallis
ANOVA, P,0.05, followed by multiple comparisons). Across all
clades, prosome length ranged from 1.17 to 1.57 mm, with a
global average of 1.32 mm (Table 3). Mean prosome length in
clade E was 1.47 mm (Table 3). Preliminary qualitative observa-
tions made on digital images of animals from four clades (A, C, G,
E) also suggest that there may be differentiation between genetic
clades in characters associated with the genital double-somite
(Fig. 5). Variation was noted in the shape and location of the
pigment spot on the genital somite (in ventral view), the shape of
the genital boss in lateral view, and the presence/absence of the
marked groove on the left side of the genital double-somite (ventral
view; Fig. 5). Clades A and C appeared to be very similar
morphologically, and both had traits characteristic of P. piseki. In
these clades, the pigment spot at the copulatory pore was large and
round, and both had a marked groove on the left side of the genital
double-somite at the posterior margin that is a key character for
identification of P. piseki [57]. Our initial observations suggest that
Clade G may be distinct from other clades with P. piseki-like
characters. Clade G animals had a large genital pigment spot, as in
clades A and C, but it was located very distally on the genital boss
(at the margin) and was distinctly oval, oriented left-right, rather
than round in shape. Animals in this clade also lacked the
characteristic groove on the left side of the genital double-somite
(Fig. 5). The genital boss in right lateral view also appeared more
elongate and less rounded than in Clades A and C. Finally, clade E
was distinctly ‘gracilian’ in genital characters, with the traits visible
in figure 5 largely as described for P. gracilis. The shape of the
genital double-somite appeared as an up-side-down-heart in
ventral view and in right lateral view the genital boss was quite
thick even towards the anterior end: both traits were illustrated by
Steuer [42] for his Forma ‘maxima’. Finally, the location of the
pigment spot on the lateral prosome was invariant, and occurred
on the right side in all animals.
Functional mtCOII Genes
cDNA and gDNA sequences obtained from the same animals
using primer sets PLPICOIIFE – PLPICOIIRC (cDNA) and
PLPICOIIFC – PLPIATP8R1 (gDNA) matched in all 17
specimens, demonstrating that the primer set used to obtain data
for the phylogeny, PLPICOIIFC – PLPIATP8R1, consistently
amplified the functional mtDNA gene copy. The majority of these
specimens belonged to clade A (8 haplotypes, 15 specimens), one
was placed in clade F, and one in clade K, confirming that all
three of these clades represent real taxonomic entities within the P.
piseki species complex and are not NUMT clades (Fig. 6).
Additional evidence that the phylogeny primers were targeting
mtDNA came from the presence of a stop codon at the end of the
mtCOII gene in sequences generated from direct sequencing (1
exception, not included).
In a small number of PCRs (7%), the phylogeny PCR primer
pair (PLPICOIIFC and PLPIATP8R1) resulted in double-banded
products that were apparent on agarose gels and these products
were not sequenced. In order to determine clade membership of
specimens yielding multiple products, we selected eight of these
samples and amplified a 376-bp PCR product (within mtCOII)
using the forward phylogeny primer PLPICOIIFC and a reverse
primer that targeted the 39 end of mtCOII. Phylogenetic analysis
of these sequences placed all eight in sub-group B2 (Fig. S3; [75]).
Multiple NUMT sequences were found in some animals from
which PCR products were cloned, and non-functional gene copies
were identifiable by high rates of divergence and long branch
lengths (Text S1, Fig. S3).
Geographic Structure
Clades in the P. piseki – P. gracilis species complex ranged from
being circumglobal in distribution to occurring at only a single site.
Clades A and B were cosmopolitan, and occurred broadly
throughout subtropical and tropical waters in all three major
ocean basins (Fig. 7). The majority of specimens sampled in the
North Atlantic were from clade A (3 sites), and this clade also
dominated our material in the North Pacific (6 sites) and western
Indian Ocean (4 sites; Fig. 7). Sample sizes for clade A specimens
ranged between 6 and 22 individuals, with an average of 15
specimens per site (Table 1). Clade A was conspicuously absent
from the South Pacific, though sample sizes were quite high there,
and this clade also was apparently absent from the South Atlantic.
Clade B occurred throughout the Pacific Ocean in subtropical and
tropical waters, and also was present in the North Atlantic and
Indian Oceans (Fig. 7). Clades F and G had Indo-Pacific
distributions, and both genetic clades were absent from the
Atlantic Ocean (Fig. 8). Clade G appeared to be more restricted to
tropical and equatorial waters than clade F, which also occurred in
subtropical waters of both the Pacific and Indian Oceans. The
majority of clade C specimens were found in the South Pacific
(Fig. S1; Table 1). Clade E was found exclusively in the southern
transition zone in the Indian, Pacific, and Atlantic Oceans (Fig.
S1). Clade J occurred in the Indian, South Pacific, and North
Atlantic Oceans, although it appeared to be quite rare throughout
its global range (Fig. S2). Only a few specimens were sampled from
each of the remaining 5 clades, and they were located either in the
North Pacific (clades D, K), Indian Ocean (clades I, L), or in the
South Pacific near the equator (clade H, Figs. S1 & S2). A
contingency analysis confirmed that the distribution and frequency
of major clades among ocean basins was non-random (x
2
= 1127;
P,0.0001).
Table 3. Summary statistics of prosome lengths (PL)
measured on genotyped P. piseki - P. gracilis specimens.
Clade N
Mean PL Std Dev PL Max PL Min PL
(mm) (mm) (mm) (mm)
A 203 1.32 0.07 1.57 1.20
B 68 1.33 0.08 1.50 1.20
C 89 1.32 0.04 1.43 1.20
E 12 1.47* 0.11 1.60 1.30
F 42 1.30 0.04 1.40 1.20
G 69 1.34 0.04 1.43 1.23
I 6 1.23 0.08 1.37 1.17
J 9 1.29 0.03 1.33 1.23
Clades are defined in Fig. 2.
*clade E has significantly larger median PL compared to all clades except G
(Kruskal-Wallis, multiple comparisons, P,0.05).
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In the hierarchical AMOVA for clade A, a significant fraction
of the variance was related to struc ture among ocean basins
(Table S1, 10.5% , P,0.0001). There was no significant variation
explained by the among localities component, consistent with
pairw ise h
ST
comparisons among all sampling sites (Table S2).
Howev er, some significant variation was see n within the North
Pacific, between ACEASIA-08 and two other North Pacific
locat ions: ACEASIA-14 (h
ST
=0.15, P,0.05) and S230-037
(h
ST =
0.10, P,0.05, Table S2). Pairwise h
ST
values between the
Indian and North Pacific Oceans ranged between 0 and 0.23,
with an average of 0.10. Similar values were found between the
North Pacifi c and North Atlantic Oceans (h
ST
range: 0.03–0.22,
average –0.12). No popula tion structure was observed betwee n
the Indian and North Atlan tic Oceans (h
ST
range: 20.03–0.04,
Table S2).
Discussion
As described in the introduction, the systematics of the genus
Pleuromamma has been treated as though it is well resolved, and
females of these species are thought to be quite straightforward to
identify. A substantial ecological literature exists on Pleuromamma
piseki and P. gracilis that relies on the existing morphological
taxonomy, with detailed reports of their horizontal and vertical
distributions [43,44], spatial and temporal patterns of abundance
[45,50], population structure [76], trophic ecology [48], and
physiological condition [51], among other topics [77]. Notably,
relatively little ecological niche separation was observed between
these two described species in a number of studies (e.g., [48]).
However, our genetic results suggest that these described
species are part of a species complex, and consist of a mosaic
of evolutionarily divergent mitochondrial lineages that are
distributed, in many cases, sympatrically across diverse ocean
environments. Further, our morphological observations, although
Figure 5. Digital images of the posterior prosome and urosome of
P. piseki
–
P. gracilis
animals from four genetic clades. Animals from
(A) clade A, (B) clade C, (C) clade G, and (D) clade E are shown in ventral and right lateral views. By traditional morphological criteria, animals in (A), (B),
and (C) would be identified as P. piseki and the animal in (D) would be identified as P. gracilis. Note that clade A and C both have a large black
pigment spot at the copulatory pore, as well as a marked groove on the left side of the genital double-somite near the posterior margin (marked by
an arrow), which was included in the original species description for P. piseki (Farran 1929). Clade G (panel C) also has a large black spot at the
copulatory pore, but the spot is located at the posterior edge of the genital boss, and appears as a right-left oval rather than round in shape. Animals
from this clade apparently lack the marked groove on the left side of the genital double-somite (absence visible at the black arrow). Finally, animals in
clades E and B have genital characters that appear ‘gracilian’ (P. gracilis-like), as shown in panel D on a clade E specimen.
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preliminary, indicate that the morphological traits currently in use
to identify these species do not map clearly onto genetic clades,
and prior ecological studies may have included spectra of
genetically distinct taxa within each of these nominal species.
These observations provide one possible explanation for the lack
of appar ent ecological specialization of P. gracilis and P. piseki in
prior reports. Our global -scale sampling suggests that these new
cryptic lineages range from being common to rare, and have
Figure 6. Placement of 17 cDNA mtCOII sequences in the Bayesian phylogeny of
P. piseki – gracilis
clades A, F, and K. cDNA sequences
are marked in red font, and bold indicates haplotypes that were sampled more than once. The scale bar units are in substitutions per site.
Relationships among the major clades are shown in figure 2.
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distinct biogeographic distributions that range from being
cosmopolitan in subtropical-tropical waters to being restricted
to particular water mass types (e.g., equator ial waters of the Indo-
Pacific). Below, we first di scuss the evidence for divergent genetic
clades and their possible status as cryptic, undescribed species,
and then review the biogeographic distributions of these new
lineages.
Cryptic Species?
Twelve evolutionarily divergent mitochondrial lineages were
discovered within this Pleuromamma species complex that were well
supported in both Bayesian and maximum likelihood mtCOII
trees (Bayesian posterior probability values ,0.7, Fig. 2), with
unambiguous reciprocal monophyly seen among all 12 clades in
the Bayesian gene tree. Six of these clades were supported with
Bayesian posterior probability values of 0.99 or higher (clades C,
E, G, H, I, L). Evolutionary divergence values between clades (5–
17% K2P distance) were high compared to within-clade diver-
gences (average –1.5%, Table 2), and were broadly comparable to
values seen between copepod species in other groups, in particular
for the most divergent clades (e.g., [34,61,78,79]). For example,
evolutionary divergences of .4–25% at mitochondrial protein-
coding genes in several marine crustaceans have been considered
to be evidence of the existence of cryptic species (e.g., [80–82]).
Supporting patterns of divergence at nuclear loci or in geography
and morphology provided additional support for the inference that
these lineages were reproductively isolated. Emerging evidence
from other planktonic calanoids also suggests that recently
divergent species can be separated by as few as 6–7 bp
substitutions at this mitochondrial gene region, with clear
concordance found among results from nuclear microsatellite
and mitochondrial markers [30]. We also validated several
features of the topology of our mtCOII tree by comparing cDNA
sequences of mtCOII derived from RNA transcripts with gDNA
sequences of mtCOII from the same individuals. Matching
sequences in all specimens confirmed the validity of clades A, K,
and F, and based on sequence properties in all other clades, we
infer that all 12 mtDNA clades reflect mitochondrial evolution and
Figure 7. Distribution map for cosmopolitan clades A (top panel) and B (bottom panel). The number of specimens (n) for each site is
given in parentheses beside the symbol. Small, open circles indicate sample locations that did not include any specimens of this clade (absence).
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are unlikely to be NUMT clades. These results all suggest that the
divergent mitochondrial lineages found within P. piseki and P.
gracilis likely reflect cryptic species. However, alternative explana-
tions are possible, and given the absence of data from additional
nuclear markers in this system, our results based on mtCOII are
preliminary.
A second possible explanation for the observation of deeply
divergent mtDNA lineages is that they are present within a single
interbreeding species (or two species, as described). The large
population sizes of marine holoplan kton would be expected to
slow the lineage sorting process, reducing the probability or rate
of loss of ancient lineages within species [37,83]. A few neritic
and/or benthic copepod species also have ver y divergent
mitochondri al lineages that are capable of interbreeding in
laboratory crosses (in some cases with lower F2 hybrid fitnes s, e.g.
[84–86]) and these complexes are used as model systems for
understanding the genomic basis of speciati on (in transition
between populations and species). Furthermore, a number of
studies have shown discordance betwe en mitochondrial and
nuclear markers in detecting either species boundaries or
population structure (e.g., [39,87,88]), and in combination, these
studies all indicate caution in relying on one or few genetic
markers for inferring the presence of dis tinct species. We
recognize the complexity of accurately inferring species bound-
aries: Therefore, we hypothesize that the mtCOII lineages within
the Pleuromamma piseki – P. gracilis speci es complex represen t
distinct species, and note that further work is req uired to validate
the appropriate systematic position of these ancient mtDNA
linea ges. In the discussion below, we proceed with the expe cta-
tion that many of these lineages are likely to be undescribed,
cryptic species, while recognizing that this inference requires
validation in subsequent work.
One of the most unexpected aspects of our results was the
observation that the morphological characters on the genital
double-somite that are currently in use to distinguish P. piseki and
P. gracilis appeared to be relatively uninformative with regards to
likely species boundaries. Animals with P. piseki-like characters
were found within five clades, with only three of these exclusively
Figure 8. Distribution map for Indo-Pacific clades F (top panel) and G (bottom panel). The number of specimens (n) for each site is given
in parentheses beside the symbol. Small, open circles indicate sample locations that did not include any specimens of this clade (absence).
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containing animals with P. piseki morphological traits (clades A, J,
C). The remaining two clades contained animals with both P. piseki
and P. gracilis morphological characters (clades B, F), indicating a
mixture of morphotypes within these clades. Comparable results
were observed for P. gracilis animals in that they sometimes co-
occurred with P. piseki within well-resolved phylogenetic clades
(e.g., clade B). These animals were identified to species based
primarily on traits associated with the genital double-somite (e.g.,
pigment spot near the copulatory pore, presence of a marked
groove on the left side of the somite), and our results suggest that
these taxonomic characters are likely insufficient for distinguishing
true species in this group. This finding is uncommon for copepods.
To our knowledge all prior examples of cryptic species complexes
have found broad congruence with existing morphological species
descriptions, but with greater diversity within each morphologi-
cally defined species (e.g., [34]). This result has significant
implications for the existing literature on P. piseki and P. gracilis,
as it suggests that it may not have been possible to accurately
identify true species using the described morphological characters
in prior work on these species.
Prior systematic and biogeographic studies in the genus
Pleuromamma have provided some suggestion of unresolved
diversity within the group. Notably, Steuer [42] described three
distinct forms within P. gracilis s. l., Forma minima, Forma maxima
and Forma Piseki. Forma Piseki is now recognized as the
contemporary species P. piseki [55], whereas Steuer’s Forma have
largely fallen out of use in the current literature. Our results
suggest that there is in fact a larger-bodied species with P. gracilis-
like morphological characters that inhabits the southern transition
zone of all ocean basins (1.3–1.6 mm PL for adult females; Table 3,
Fig. S1). This lineage, clade E in our material, is largely congruent
with Steuer’s observations of Forma maxima, with the exception
that we find no evidence that clade E is distributed outside of the
southern transition zone region. The morphology of the genital
boss of our clade E specimens (Fig. 5D) shows the up-side-down
heart shape seen in ventral view for P. gracilis Forma maxima
(Steuer 1932, his Textfig. 134), and is also thicker in right lateral
view than as described for Forma minima (compare our figure 5D
to Steuer Textfig. 134 and 135). Therefore, following more
extensive examination of clade E specimens, it may be appropriate
to elevate Forma maxima to species. We also anticipate that
Pleuromamma gracilis s.s. will correspond to genetic clade B of this
study, once a thorough systematic revision has been completed.
Resolving the position of P. borealis, the only remaining described
small-bodied species in the genus, is also a high priority for future
work. Our preliminary morphological observations on genital
characters suggest that these traits will continue to be informative
within the genus (e.g., see Fig. 5C), although the specific traits
currently in use as taxonomic characters may not be definitive for
identification to species. The systematic identity of the name-
bearing lineages for described species P. piseki and P. gracilis
remains to be verified, and will be challenging given broad
sympatry of many of the undescribed lineages in the North
Atlantic. The type locality for P. piseki was not specifically
identified, although its distribution was listed as the North
Temperate and Tropical Atlantic from 29u109N southwards as
well as off New Zealand [57], and P. gracilis was first described
from Messina in the Mediterranean Sea [89]. Five genetic clades
co-occur in these areas (North Atlantic; A, B, C, E, J; Table 1). It is
unclear which of these may correspond to the material originally
examined for species descriptions. Finally, there may be addi-
tional, undetected diversity within this species complex: a
number of the divergent lineages discovered here were found in
only a few individuals, suggesting that the global ocean remains
under-sampled.
Biogeography of Cryptic Lineages
The novel genetic lineages within P. piseki and P. gracilis appear
to be ecologically divergent, with distinct biogeographic distribu-
tions across varied pelagic habitats. Among the 12 genetic clades,
some were common in our material, and occurred at nearly half
the collection sites (e.g., clade A), while others were rare, and
collected at only a single sampling site (e.g., clades H, I, and L).
The biogeographic distributions of common lineages therefore can
be interpreted from the present material, while rare clades remain
poorly characterized in terms of their distributions across pelagic
habitats. Four clades had circumglobal distributions (clades A, B,
C, and J) and were broadly sympatric across subtropical and
tropical waters, indicating a capacity to thrive across a range of
pelagic habitat types from broadly eutrophic to oligotrophic
systems (e.g., subtropical gyres, equatorial provinces). These clades
also were the most numerous at many of our sampling sites (in
particular clades A, B). Two clades, F and G, were Indo-Pacific in
distribution, and occurred sympatrically with each other as well as
with the cosmopolitan clades. Although clade F was quite
widespread across oligotrophic and eutrophic waters in the Pacific
and Indian Oceans, clade G appeared to be largely restricted to
equatorial waters, a distributional type that is common to many
other zooplankton species (e.g., Clausocalanus minor, [90], Subeuca-
lanus subcrassus, [19], Euphausia diomediae, [91]). We also found that
many of the divergent clades occurred in only one or two locations
within the same ocean basin (e.g. North Pacific, clades D, K), and
sometimes consisted of only a single individual from our material
(e.g., clade L). These observations suggest that the global diversity
within the small-bodied Pleuromamma species is likely under-
sampled in this study. Finally, although the branching topology
of our mtCOII phylogeny was not well resolved at most deeper
nodes within the tree (Fig. 2), widespread clades do not appear to
be close relatives to one another, and in the cases where sister
relationships were well-supported (e.g., Clades C, L), a wide-
spread, common clade was most closely related to a lineage that
was rare or restricted in distribution.
Conclusions
Discovery of cryptic species is common, in particular for marine
invertebrate groups (e.g., [31,92]). However, an inability to
identify common, reproductively isolated species can impede
understanding of ecosystem dynamics, making discovery and
description of cryptic species complexes an important endeavor.
Pleuromamma is the most ecologically significant genus of the diel
vertical migratory (DVM) zooplankton assemblage, due to the
high in situ abundance and global distribution of members of this
genus, as well as their extensive vertical migrations, which result in
active export of carbon and nitrogen from surface waters into the
deep ocean. Members of this genus, including P. gracilis and P.
piseki, have been reported as numerical dominants of the
zooplankton assemblage in surface waters at night [45]. Results
reported here demonstrate that extensive cryptic diversity exists
within smaller members of this common copepod genus, with
broad sympatry of .5 divergent mtDNA lineages in most regions
of the global ocean. We hypothesize that many of these mtDNA
lineages are undescribed, cryptic species, and call for formal
validation of the systematic position of these lineages through
further work on morphological and genetic variation in this species
complex. These putative new species show evidence of ecological
divergence, with distinct biogeographic distributions across water
Pleuromamma Cryptic Species Complex
PLOS ONE | www.plosone.org 15 October 2013 | Volume 8 | Issue 10 | e77011
mass types. Formal description of these new species will be
important to detecting long-term trends in abundance of the
dominant species in marine zooplankton communities, resolving
their population responses to climate forcing, and inferring their
impact on biogeochemical cycling in the upper ocean.
Supporting Information
Figure S1 Distribution map for clades C, D, I and E. The
number of specimens (n) for each site is given in parentheses beside
the symbol. Small, open circles indicate sample locations that did
not include any specimens of these clades (absence). Colors and
symbols for each clade as indicated in the legend.
(TIF)
Figure S2 Distribution map for clades J, K, L, and H. The
number of specimens (n) for each site is given in parentheses beside
the symbol. Small, open circles indicate sample locations that did
not include any specimens of these clades (absence). Colors and
symbols for each clade as indicated in the legend.
(TIF)
Figure S3 Bayesian phylogeny of P. piseki – P. gracilis clades B
and F, incorporating sequences from cloning experiments and
other problematic data from genomic DNA amplifications. (A)
Bayesian phylogeny of clade B, (B) Bayesian phylogeny of clade F.
Green text = sequences cloned from genomic DNA amplifications.
Blue text = mtCOII amplifications (376-bp) with primers PLPI-
COIIFC & COIIR10 that were used to determine the clade
membership of animals yielding multiple products in PCR.
NUMTs (premature stop codon) are highlighted in grey. Bold
text indicates haplotypes that were sampled more than once. The
scale bar units are in substitutions per site.
(TIF)
Table S1 Population structure within clade A, across the Indian,
North Pacific, and North Atlantic Oceans. Results from a
hierarchical analysis of molecular variance (AMOVA).
(DOCX)
Table S2 Pairwise h
ST
values between sampling sites within
clade A. Significant values (P-value ,0.05) are shown in bold.
The Q-value was ,0.05 for all P-values ,0.05. Indian Ocean
sites = VANC-02, VANC-06, VANC-09, VANC-11; North Pacific
sites = ASIA-14, ASIA-08, HOT, S226-48, S230-37, STAR-49;
North Atlantic sites = TRAN, MP3-12, MP3-14.
(DOCX)
Text S1 Supplementary Text on characterizing NUMTs.
(DOCX)
Acknowledgments
We thank D-W Jung, J. Ayau, S. Chang, V. Flynn, J. Bernier and E.
Portner for assistance in the laboratory. We also thank R. Harmer for
plankton collections on the AMT20 cruise (in 2010). Janet Grieve, Katja
Peijnenburg, and one anonymous reviewer provided thoughtful comments
on the manuscript.
Author Contributions
Conceived and designed the experiments: KH EG DBC. Performed the
experiments: KH EG. Analyzed the data: KH EG DBC. Contributed
reagents/materials/analysis tools: EG. Wrote the paper: KH EG DBC.
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