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Article

Distinguishing Long-Discussed Cryptic Species of the Epibiotic Goose-Neck Barnacle of the Genus Conchoderma (Thoracicalcarea: Lepadidae) with Integrative Taxonomy

by
Benny K. K. Chan
1,* and
Yu-Hsuan Chen
1,2
1
Biodiversity Research Center, Academia Sinica, Taipei 11529, Taiwan
2
Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei 10663, Taiwan
*
Author to whom correspondence should be addressed.
Diversity 2022, 14(8), 593; https://doi.org/10.3390/d14080593
Submission received: 22 June 2022 / Revised: 18 July 2022 / Accepted: 20 July 2022 / Published: 24 July 2022
(This article belongs to the Topic Arthropod Biodiversity: Ecological and Functional Aspects)
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Abstract

:
Naked goose neck barnacles Conchoderma can grow on a wide variety of marine organisms. The taxonomic status of two of its species—C. virgatum and C. hunteri—are currently controversial. Some studies suggest that C. hunteri is a subspecies, variety or growth forms of C. virgatum, because both have great morphological variations, but other studies consider C. hunteri and C. virgatum to be distinct species. The present study examines the morphology and sequence divergence of the COI gene in C. virgatum, C. hunteri and other closely related species. There are consistent morphological differences between C. virgatum and C. hunteri in the tergum, carina and fifth teeth of the mandible. Phylogenetic analysis based on the divergence in the COI gene revealed that C. virgatum and C. hunteri form sister clades with high bootstrap values. The K2P distances within C. hunteri and C. virgatum are 0.034 ± 0.008 and 0.002 ± 0.001 for the COI sequences, respectively. The K2P distance between C. hunteri and C. virgatum is 0.097 ± 0.016. Morphological and molecular evidence confirm that C. hunteri is a valid species.

1. Introduction

Marine biologists have been interested in the naked goose neck barnacles Conchoderma (Lepadidae; latest classification see [1]) for a long time, because species in the genus live on a variety of marine organisms, including copepods, isopods, sea snakes, whales, fishes, crabs and sea turtles [2,3,4,5,6]. The capitulum of Conchoderma has highly reduced shell plates, including the tergum, scutum and carina [7,8]. At present, Conchoderma comprises four or five species: C. auritum, C. virgatum (Spengler, 1790) [9], C. hunteri (Owen, 1830) [10], C. chelonophilum (Leach, 1818) [11] and C. indicum (Daniel, 1953) [12]; however, the taxonomic status of C. virgatum and C. hunteri is controversial because they have great morphological variations in their tergum, scutum and capitulum [13,14].
C. virgatum has three longitudinal bands on its capitulum [7,15] (Figure 1). C. hunteri has a brown colored capitulum and occasionally with faint vertical bands [7] (Figure 1). The number of filamentary appendages, morphology of cirri and mouth parts are similar between C. hunteri and C. virgatum [15]. Darwin [7] described C. hunteri and compared it to C. virgatum with the comment (page 155) “I have very great doubt whether I have acted rightly in considering this as a species.” However, based on the variation in the morphology of their tergum, Darwin [7] concluded C. hunteri and C. virgatum are different species. Hoek [16] considered C. hunteri to be morphologically similar to C. virgatum chelonophilum and C. hunteri to be a subspecies of C. virgatum. Annandale [17] followed Hoek’s subspecies designation for C. hunteri. Hiro [13] doubted that C. hunteri is a sub-species or variety of C. virgatum or a juvenile form of C. virgatum, because previous records of C. hunteri identified the species to be smaller than C. virgatum. Finally, Hiro [13] considered C. hunteri to be a subspecies of C. virgatum. Zevina [18] supported C. hunteri and C. virgatum as conspecific. In contrast, Liu and Ren [8,19] and Yamato et al. [4] considered C. virgatum and C. hunteri to be different species. Yorisue et al. [5] doubted the taxonomic status of C. virgatum and C. hunteri when examining the diversity of Conchoderma in crabs in Japan and called the two species Conchoderma sp. in their study.
To ascertain the taxonomic status of C. hunteri and C. virgatum, it is essential to conduct further detailed morphological comparisons and molecular studies on their sequence divergences. The present study examines the morphology and sequence divergence in the COI gene of C. hunteri and C. virgatum to determine the taxonomic status of C. hunteri and C. virgatum.

2. Materials and Methods

2.1. Sample Collections

A colony of Conchoderma virgatum was collected in Kenting, Taiwan. Several colonies of C. hunteri were collected in Taiwan and the Philippines (see materials examined below for the collection details).

2.2. Morphological Examination

The size (mm) of the stalked barnacle was measured as capitular length (CL) by capitular width (CW). Specimens were dissected under a Leica stereomicroscope, mouth parts, setal-types of cirri and dwarf males were examined under a Zeiss Axioplan compound microscope installed with objectives APO 40× and APO 20× lenses.

2.3. Molecular Analysis

The mitochondrial COI gene (cytochrome c oxidase subunit I) was sequenced from Conchoderma specimens. DNA was extracted from muscle tissue using the commercial QIAamp Tissue Kit (QIAGEN). Partial sequences of the mitochondrial COI gene were amplified using a PCR universal primer set (LCO-1490/HCO-2198) for DNA barcoding [20]. Polymerase chain reaction (PCR) was performed using 25 μL of a solution containing 12.5 μL of Taq 2× Tools Taq Master Mix (AMPLIQON), 7.5 μL of deuterium-depleted water, 1 μL of each primer (5 µM) and 3 μL of the DNA template. The thermal regime entailed initialization for 4 min at 94 °C, denaturation using 35 cycles for 0.5 min at 94 °C, 0.5 min at 50 °C and 1 min at 72 °C, elongation for 10 min at 72 °C and a final extension at 12 °C. The PCR products were observed on 1.5% agarose gel, and the most intense products were used for Sanger sequencing. Successfully amplified PCR products were purified and sequenced using Genomics BioSci & Tech Ltd. Sequences were deposited in National Center for Biotechnology Information (NCBI) GenBank (https://www.ncbi.nlm.nih.gov/) and Barcode of Life Data system (BOLD, https://www.boldsystems.org/).

2.4. Phylogenetic Analysis

Eleven COI sequences of species in Lepadidae (KU204318, GU993634, MT563438, MT563441 and KU204353); Heteralepadidae (EU884174 and EU8841690); Poecilasmatidae (KC138499, KC138498 and EU884173) and Scalpellidae (KT208441) were downloaded from NCBI GenBank and included in the dataset. We also included 1 COI sequence of Lepas (ON938319) and Oxynaspis (ON938312) from our specimen collection. All the sequences were aligned with Clustal W in MEGAX [20,21,22] (See Supplementary Materials for the aligned sequences).
Neighbor-joining (NJ) and maximum likelihood (ML) tree were inferred by using MEGAX [23]. A Bayesian inference (BI) analysis was also performed using MrBayes 3.2.7 [24]. For the NJ and ML tree, the T92 + G model was selected as the best substitution model according to the model test in MEGAX with 1000 bootstrap replicates [25]. The BI analysis was conducted with 1 × 106 generations of 5 MCMC chains. Trees were saved every 1000 generations, and the first 25% of trees were discarded as burn-in.
Assemble species with automatic partitioning analysis (ASAP) (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html, accessed on 11 July 2022) [26], Poisson tree processes (PTP) model (https://species.h-its.org/ptp/, accessed on 11 July 2022) [27] and Generalized Mixed Yule Coalescent (GMYC) method [28,29] were used to confirm the species delimitation. For ASAP, Juke-Cantor model (JC69), Kimura 2-parameter model (K2P) and Simple Distance (p-distances) were used, respectively. The PTP method was performed with the ML tree (see above) for 500,000 MCMC generations and saved every 500 generations. The first 25% of generations were discarded as burn-in. For GMYC method, phylogenetic tree was inferred by using BEAST v. 1.10.4 [30] with Yule process. HKY + G was selected as substitution model based on the result of model test in MEGAX. Three MCMC chains were run for 1 × 106 generations, and sampled every 1000 generations. The run convergence was visually verified in Tracer v. 1.7.2 [31], and the effective sample size (ESS) of all parameters was >200. The tree files from the 3 runs were combined and discarded the first 25% as burn-in using LogCombiner v. 1.10.4(BEAST package) and assessed using TreeAnnotator v. 1.10.4 (BEAST package). After ultrametric tree was constructed, the GMYC method was performed in R software v. 4.1.2 using the “splits” package (Species Limits by Threshold Statistics) v. 1.0-20 [32]. Kimura 2-parameter (K2P) distances of COI sequences within/between species were also calculated using MEGAX [33] (Table 1).

3. Results

3.1. Taxonomy

Superorder Thoracicalcarea Gale, 2015 [39].
Order Scalpellomorpha Buckeridge and Newman, 2006 [40].
Family Lepadidae Darwin, 1851 [7].
Genus Conchoderma von Olfers, 1814 [41].
Conchoderma virgatum (Figure 1A, 2–6).
Lepas virgata Spengler 1790: Figure [9].
Conchoderma virgata Darwin 1851: 146, pl. 3, Figure 4 [7].—Zevina 1980: 692 [42].
Conchoderma virgatum Hoek 1883: 55 [16].—Gruvel 1905: 144, Figure 169 [43].—Gruvel 1920: 38 [44].—Pilsbry 1907: 99, pl. 9, Figure 1 [15].—Annandale 1909: 80 [17].—Stebbing 1910: 566 [45].—Jennings 1918: 59 [46].—Nilsson-Cantell 1921: 242 [47].—Nilsson-Cantell 1928: 16, Figure 7 [48].—Nilsson-Cantell 1930: 251, pl. 1, Figure 4 [49].—Nilsson-Cantell 1939: 236 [50].—Broch 1924: 58, Figure 20 [51].—Barnard 1924: 61 [52].—Krüger 1927: 13 [53].—Hiro 1936: 623, Figure 2 [54].—Hiro 1937a: 402, Figure 5 [13].—Hiro 1937b: 62, Figure 53 [55].—Hiro 1939a: 205 [56].—Hiro 1939b: 248 [57].—Utinomi 1970: 341 [58].—Balakrishnan 1969: 102, pl. 1 [59].—Newman and Ross 1971: 35, Figure 11, pl. 5E [2].—Hastings 1972: 274 [3].—Williams 1978: 109, pl. 1 [60].—Munroe and Limpus 1979: 198 [61].—Foster and Willan 1979: Figure 3d [62].—Zevina 1982: 27, Figure 16 [18].—Liu and Ren 1985: 225, Figure 23, 24, pl. 6: 6–11 [19].—Eckert and Eckert 1987: 682 [63].—Yamato et al. 1996: 338, Figure 2 (left) [4].—Liu and Ren 2007: 199, Figure 82 [8].—Alonso et al. 2010: 167, Figure 2 [64].
Materials examined: Xiangjiaowan, Kenting, Pingtung Co., June. 2003: 9 specimens (CL 4.26~9.35 mm, CW 2.21~6.01 mm) (CEL-BB79).
Diagnosis: Capitulum with three or more dark longitudinal bands. Tergum not bended, mandible with five distinct large teeth.
Description:
Capitulum. Capitulum flattened, light blue in color when alive, five extremely reduced opercular plates (a single carina, paired scutum and tergum; Figure 1A), three dark longitudinal purplish brown stripes extending from top of capitulum to the base of peduncle (Figure 1A). Head region of capitulum rectangular or trapezium-shaped (Figure 1A). Tergum straight, distal end pointed, proximal end blunted (Figure 1A). Carina sinus and short, tapers to each end. Scutum tri-lobed and y-shaped, upper and lower lobes narrow, medial lobe wide and bunt triangular-shaped (Figure 1A).
Cirri. Cirrus I, anterior rami six segmented, posterior rami eight segmented, segments protuberant (except the first-third distal segments) with height 3 times greater than width (Figure 2A–D), bearing dense simple and serrulate-type setae (Figure 2B–D). Base of cirrus I with two pairs of filamentary appendages (Figure 2A). Cirrus II, posterior rami 10-segmented, anterior rami 8-segmented, up to last proximal 5 segments protuberant, with denser serrulate setae (Figure 2E–G). Intermediate segments of cirrus II with two pairs of long serrulate setae and two pairs of short simple setae (Figure 2H). Base of cirrus II without filamentary appendages. Cirrus III, posterior ramus 10 segmented, anterior ramus broken (Figure 3A), intermediate segment bears two pairs of long and two pairs of short simple setae (Figure 3B,D). Distal segments with robust simple setae (Figure 3C). Cirrus IV–VI similar in morphology, both anterior and posterior ramus similar in length (Figure 3E–H and Figure 4A–F). Intermediate segments of cirrus III to VI bears 2 pairs of long simple setae and one pair of short simple setae (Figure 3H and Figure 4B,F). Base of each cirrus III–VI bear one pair of filamentary appendages. Penis thick and short, without basidorsal point, being 1/4 length of the cirrus VI (Figure 4G,H).
Trophi. Maxilla circular, with simple and serrulate-type setae on exterior margin (Figure 5A–C). Maxillule with one large setae at upper margin (Figure 5D,E), cutting edge divided into three zig-zagged portions (Figure 5D), each portion three to four large setae (Figure 5F,G). Mandibles penta-dentoid (Figure 6A); first teeth large, robust and sharp, separated from the remainders (Figure 6B). Second and fifth teeth sharp, large and robust (Figure 6B,C). Teeth surfaces with small sharp triangular setae. Inferior angle with two small spines (Figure 6D). Mandibular palp elongated and with serrulate setae on tip and outer margin (Figure 6E,F). Labrum not bullate, with a small, concaved notch; a single array of small sharp teeth at the notch (Figure 6G,H).
Distribution: Cosmopolitan. On surfaces of buoys, snakes, sea turtles, copepods, isopods, fishes.
Conchoderma hunterFigure 1B,C, 7–12.
Cineras hunteri Owen 1830: 71 [10]
Conchoderma hunteri Darwin 1851: 153, pl. 3, Figure 3 [7].—Gruvel 1905: 145, Figure 169 [43].—Barnard 1955: 247 [65].—Liu and Ren 1985: 227, Figure 25, pl. 6, 12–16 [19].—Yamato et al. 1996: 338, Figure 2 (right) [4].—Liu and Ren 2007: 200, Figure 83 [8].
Conchoderma virgatum var. hunteri Annandale 1909: 82 [17].—Krüger 1911: 26, pl. 3, Figures 20–22 [66].
Conchoderma virgatum var. japonica Krüger 1911: 27, pl. 3, Figure 23 [66].
Conchoderma virgatum forma hunteri Broch 1931: 28 [67].—Nilsson-Cantell 1938: 27 [68].
Conchoderma virgatum hunteri Hiro 1937a: 402, Figure 6 [13].—Hiro 1937b: 63, Figure 53 [55].—Hiro 1939a: 205 [56].—Utinomi 1968: 167 [69].—Utinomi 1970: 341 [58].—Gordon 1970: 21, Figure 6 [70].—Dong et al. 1980: 125 [71].
Materials examined: CP2362, Panglao Island, the Philippines, 8°56.5′ N, 123°32.7′ E, 679–684 m, 6 May 2005: 30 specimens (CL 10.56~22.64 mm, CW 6.9~10.56 mm). Tiaoshi, Kenting, Pingtung Co., 2 Feb. 2007: 7 specimens (CL 14.86~19.4 mm, CW 9.29~12.52 mm) (CEL-BB94).
Diagnosis: Capitulum dark brown or purplish brown, tergum bended at 1/3 distal portion. Mandible with five teeth, the fifth teeth small.
Description:
Capitulum. Capitulum flattened, dark brown or purplish brown in color, opercular plates extremely reduced, occasion with dark stripes extending from top of capitulum to the peduncle and base (Figure 1B,C). Upper margin of capitulum pointed. Tergum bended at one fourth of its distal length (Figure 1B), with one end pointed and the other end blunted. Carina long, overlapping with tergum at 1/4 distal region. Scutum tri-lobed (Figure 1B,C).
Cirri. Cirrus I, posterior and anterior rami eight segmented, segments protuberant (except the first-third distal segments) with height three times greater than width (Figure 7A–D), bearing dense simple and serrulate-type setae (Figure 7B,C). Base of cirrus I with two pairs of filamentary appendages (Figure 7A). Cirrus II, posterior rami 12-segmented, anterior rami 11-segmented, up to the last proximal 5 segments protuberant, with denser serrulate setae (Figure 7E–G). Intermediate segments of cirrus II with dense serrulate and simple setae (Figure 7H). Base of cirrus II without filamentary appendages. Cirrus III, posterior ramus 16-segmented, anterior ramus 15-segmented (Figure 8A), intermediate segment bears two pairs of long and two pairs of short simple setae (Figure 8B,D). Distal segments with robust simple setae (Figure 8C). Cirrus IV–VI similar in morphology, both anterior and posterior ramus similar in length (Figure 8E–H and Figure 9A–F). Intermediate segments of cirrus III to VI bears two pairs of long simple setae and one pair of short simple setae (Figure 8H and Figure 9B,F). Base of each cirrus III–VI bear one pair of filamentary appendages. Penis thick and short, without basidorsal point, being 1/4 length of the cirrus VI (Figure 9G,H).
Trophi. Maxilla circular, with simple and serrulate-type setae on exterior margin (Figure 10A–C). Maxillule with one large setae at upper margin and cutting edge divided into three zig-zagged portions, each portion three to four large setae; interior margin slightly convex (Figure 10D–H). Mandibles penta-dentoid; first teeth large, robust and sharp, separated from the remainders (Figure 11A–D). The fifth teeth are very small, 4–5 times smaller than the third and fourth teeth (Figure 11C,D). Size of fifth teeth comparable to the size of the inferior angle (Figure 11D). Mandibular palp elongated and with serrulate setae on tip and outer margin (Figure 11E,F). Labrum not bullate, with a small, concaved notch; a single array of small sharp teeth at notch (Figure 11G,H).
Distribution: Cosmopolitan.
Remarks: There are consistent morphological differences between C. virgatum and C. hunteri. The bending of the tergum and size of the fifth teeth of the mandible are consistently diagnostic between the two species. C. hunteri has a bended tergum, and its fifth tooth in the mandible is much smaller than that of C. virgatum (Figure 12).

3.2. Molecular Analysis

All the phylogenetic results suggested that both Conchoderma hunteri and C. virgatum were clustered into their own clades with high bootstrap values and posterior probabilities support, except for the BI, which the posterior probability for C. virgatum cluster was 67.43 (Figure 13). Both species form a sister group and C. auritum has the sister relationship with C. hunteri and C. virgatum in the NJ and ML results (Figure 13). However, the BI analysis showed polytomy with C. hunteri + C. virgatum, C. auritum, and L. australis + L. anserifera (Figure 13).
The K2P distances within C. hunteri and C. virgatum were 0.002 ± 0.001 and 0.034 ± 0.008 for the COI sequences, respectively. The K2P distances between C. hunteri and C. virgatum were 0.097 ± 0.016. The K2P distances between these two species and other species ranged from 0.242 to 0.354 (Table 2).
Generally, the three morphospecies of Conchoderma were always in different MOTUs (Molecular Operational Taxonomic Units). However, PTP analyses further split C. hunteri and C. virgatum into two MOTUs each.

4. Discussion

The present study revealed that Conchoderma virgatum and C. hunteri are different species based on morphological and molecular evidence, rejecting the hypothesis of their conspecificity proposed by Hiro (1937) [13] and Zevina (1982) [18]. First of all, the species have different terga. The tergum of C. hunteri bends at a 1/4 position of the distal region. The tergum of C. virgatum is relatively straight and does not bend. The distal one-third portion of the carina overlaps with the tergum in C. hunteri. Mandibles of C. hunteri have small fifth teeth, in contrast to the bigger fifth teeth in C. virgatum. There are no diagnostic differences between their somatic bodies—both have a similar number and position of paired filamentary appendages, cirri, and mouth parts (except the mandible).
Based on our molecular analysis, Conchoderma virgatum and C. hunteri form sister clades with high support, indicating that the two species are closely related, and these two species also share similar epibiotic habitats [2,3,4,5,6] and similar morphology (cirri and filamentary appendages on somatic bodies). The phylogeny analysis and specimen delimitation result of ASAP and GMYC all suggested that C. virgatum and C. hunteri are different species. The results of PTP also divide C. virgatum and C. hunteri into two groups. However, PTP further indicated that C. virgatum might have two groups, which need further examination on morphological features from larger sample sizes to confirm.
The present study confirmed that there are currently five species in Conchoderma—C. virgatum, C. hunteri, C. auritum, C. indicum, and C. chelonophilum which differ in their number of filamentary appendages, shape of their tergum, and morphology of their capitulum. Darwin [7] considered C. chelonophilum to be a variety of C. virgatum that lives exclusively on turtles. Hoek [16] redescribed C. chelonophilum and doubted that it is its own species based on its exclusively occurrence on sea turtles, smaller size, and different capitulum shape compared to C. virgatum. Due to high morphological variations between C. chelonophilum and C. virgatum, Hoek [16] considered C. chelonophilum to be a subspecies of C. virgatum. Jones [72] considered C. chelonophilum to be a distinct species. A further record of C. virgatum chelonophilum is from loggerhead turtles in Australia [61]. A more recent record of C. virgatum is from sea turtles in Uruguay [64], but that publication contained photographs of a colony of Conchoderma on the turtles without detailed morphological examinations. Those specimens from turtles in Uruguay probably represents C. chelonophilum. Moving forward, the taxonomic status of C. chelonophilum should be further evaluated using morphological and molecular analyses.
Key to species of Conchoderma
(1)
With ear-like extension in the capitulum…………………Conchoderma auritum
(2)
Without ear-like extension in capitulum…………………………………..………(3)
(3)
2nd cirri with filamentary appendages……………………Conchoderma indicum
(4)
2nd cirri without filamentary appendages………………………………………(5)
(5)
Tergum bended at 1/3 distal portion………………………Conchoderma hunteri
(6)
Tergum not bended………………………………………………….…………(7)
(7)
Live on sea turtles……………………………………Conchoderma chelonophilum
(8)
Live on other marine substratum/organism……………Conchoderma virgatum

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d14080593/s1. Aligned sequences with with Clustal W in MEGAX.

Author Contributions

Conceptualization: B.K.K.C., methodology, B.K.K.C. and Y.-H.C.; software, Y.-H.C.; validation, B.K.K.C. and Y.-H.C.; formal analysis, Y.-H.C.; investigation, B.K.K.C.; resources, B.K.K.C.; data curation B.K.K.C. and Y.-H.C.; writing—original draft preparation, B.K.K.C. and Y.-H.C.; writing—review and editing, B.K.K.C. and Y.-H.C.; visualization, B.K.K.C. and Y.-H.C.; supervision, B.K.K.C.; project administration, B.K.K.C.; funding acquisition, B.K.K.C. All authors have read and agreed to the published version of the manuscript.

Funding

BKKC is supported by a grant in Ministry of Science and Technology, Taiwan (110-2923-B-001-003-MY3).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the molecular sequences are available in Genbank and BOLD.

Acknowledgments

The PANGLAO 2005 deep-sea expedition onboard the research vessel M/V DA-BFAR was a collaboration between the Muséum national d’Histoire naturelle, Paris (MNHN, Philippe Bouchet), the National Fisheries Research and Development Institute (NFRDI, Ludivina Labe), the Philippines Bureau of Fisheries and Aquatic Resources (BFAR), the National University of Singapore (NUS), the University of San Carlos (USC), and the National Taiwan Ocean University (NTOU). The cruises were affiliated with the Census of Margins (CoMarges) component of the Census of Marine Life, and we acknowledge funding from the French Ministry of Foreign Affairs (2005 cruise) and the Total (the Philippines) Corporation. Malcolm Sarmiento, Director of BFAR, kindly made M/V DA-BFAR available, and Noel Saguil (USC Associate) organized the logistics. Thanks to Wei-Peng Hsieh (Academia Sinica) helped us with the dissections and image arrangements and Yao-Feng Tsao (Academia Sinica) helped with the molecular analysis. We thank Noah Last (The Third Draft Editing) for editing the English of the manuscript. We would like to thank the two anonymous reviewers for their constructive comments to further enhance the quality of our manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. (A). Conchoderma virgatum, showing the capitulum with three longitudinal dark bands. (B). Conchoderma hunteri, specimen with faint dark bands on the capitulum. (C). Conchoderma hunteri with pale brown (left) and darker brown (right) capitulum. Note tergum bends at the distal 1/4 portion. (D). C. virgatum, redrawn from Darwin (1851) [7]. (E). C. virgatum redrawn from Pilsbry (1907) [15]. (F). C. hunteri, redrawn from Darwin (1851) [7]. (G). C. indicum redrawn from Daniel (1953) [12]. (H). C. chenolophilum redrawn from Darwin (1851) [7]. (I). C. chenolophilum redrawn from Hoek (1883) [16]. (J). A Japanese plastic key chain of C. virgatum and with its biological descriptions. Ca—Carina, S—scutum and T—tergum.
Figure 1. (A). Conchoderma virgatum, showing the capitulum with three longitudinal dark bands. (B). Conchoderma hunteri, specimen with faint dark bands on the capitulum. (C). Conchoderma hunteri with pale brown (left) and darker brown (right) capitulum. Note tergum bends at the distal 1/4 portion. (D). C. virgatum, redrawn from Darwin (1851) [7]. (E). C. virgatum redrawn from Pilsbry (1907) [15]. (F). C. hunteri, redrawn from Darwin (1851) [7]. (G). C. indicum redrawn from Daniel (1953) [12]. (H). C. chenolophilum redrawn from Darwin (1851) [7]. (I). C. chenolophilum redrawn from Hoek (1883) [16]. (J). A Japanese plastic key chain of C. virgatum and with its biological descriptions. Ca—Carina, S—scutum and T—tergum.
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Figure 2. Conchoderma virgatum. (A) Cirrus I, showing two filamentary appendages at base region. (B) Posterior ramus, showing serrulate setae. (C) Anterior ramus, showing serrulate setae. (D) Protuberant segment of cirrus I, showing serrulate setae. (E) Cirrus II. (F) Posterior ramus showing serrulate setae. (G) Anterior ramus, showing serrulate setae. (H) Intermediate segment of anterior ramus, showing serrulate setae. Scale bars in μm.
Figure 2. Conchoderma virgatum. (A) Cirrus I, showing two filamentary appendages at base region. (B) Posterior ramus, showing serrulate setae. (C) Anterior ramus, showing serrulate setae. (D) Protuberant segment of cirrus I, showing serrulate setae. (E) Cirrus II. (F) Posterior ramus showing serrulate setae. (G) Anterior ramus, showing serrulate setae. (H) Intermediate segment of anterior ramus, showing serrulate setae. Scale bars in μm.
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Figure 3. Conchoderma virgatum. (A) Cirrus III, note single filamentary appendage at base, anterior ramus broken. (B) Intermediate segments of posterior ramus, showing serrulate setae. (C) Distal segment of posterior ramus. (D) Magnified segment of anterior ramus of cirrus III, showing serrulate setae. (E) Cirrus IV, showing small filamentary appendage at base. (F) Simple-type setae at cirrus IV. (G) Distal end of posterior ramus of cirrus IV, showing short robust simple setae. (H) Intermediate segment of cirrus IV, showing serrulate setae. Scale bars in μm.
Figure 3. Conchoderma virgatum. (A) Cirrus III, note single filamentary appendage at base, anterior ramus broken. (B) Intermediate segments of posterior ramus, showing serrulate setae. (C) Distal segment of posterior ramus. (D) Magnified segment of anterior ramus of cirrus III, showing serrulate setae. (E) Cirrus IV, showing small filamentary appendage at base. (F) Simple-type setae at cirrus IV. (G) Distal end of posterior ramus of cirrus IV, showing short robust simple setae. (H) Intermediate segment of cirrus IV, showing serrulate setae. Scale bars in μm.
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Figure 4. Conchoderma virgatum. (A) Cirrus V, with single small filamentary appendage at base, highlighted in D. (B) Intermediate segment of cirrus V. (C) Distal segment of cirrus V, showing robust simple setae. (D) Filamentary appendage at based. (E) Cirrus VI. Note one ramus was broken. (F) Serrulate setae at intermediate segment of cirrus VI. (G) Penis. (H) Apex of penis. Scale bars in μm.
Figure 4. Conchoderma virgatum. (A) Cirrus V, with single small filamentary appendage at base, highlighted in D. (B) Intermediate segment of cirrus V. (C) Distal segment of cirrus V, showing robust simple setae. (D) Filamentary appendage at based. (E) Cirrus VI. Note one ramus was broken. (F) Serrulate setae at intermediate segment of cirrus VI. (G) Penis. (H) Apex of penis. Scale bars in μm.
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Figure 5. Conchoderma virgatum. (A) Maxilla. (B) Simple setae at outer margin of maxilla. (C) Serrulate setae at inner margin of maxilla. (D) Maxillule showing three zig-zag regions at the cutting edge. (E) Large spine of maxillule. (F) Simple setae at the base of large spine of maxillule. (G) Cutting edge of maxillule. (H) Serrulate-type setae at the cutting edge of maxillule. Scale bars in μm.
Figure 5. Conchoderma virgatum. (A) Maxilla. (B) Simple setae at outer margin of maxilla. (C) Serrulate setae at inner margin of maxilla. (D) Maxillule showing three zig-zag regions at the cutting edge. (E) Large spine of maxillule. (F) Simple setae at the base of large spine of maxillule. (G) Cutting edge of maxillule. (H) Serrulate-type setae at the cutting edge of maxillule. Scale bars in μm.
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Figure 6. Conchoderma virgatum. (A) Mandible showing five teeth. (B) First and second teeth of mandible. (C) Third, fourth, and fifth teeth of mandible. (D) First teeth of mandible showing a surface with dense simple setae. (E) Mandibulatry palp. (F) Serrulate setae at the margin of palp. (G) Labrum. (H) Cutting edge of labrum showing small teeth. Scale bars in μm.
Figure 6. Conchoderma virgatum. (A) Mandible showing five teeth. (B) First and second teeth of mandible. (C) Third, fourth, and fifth teeth of mandible. (D) First teeth of mandible showing a surface with dense simple setae. (E) Mandibulatry palp. (F) Serrulate setae at the margin of palp. (G) Labrum. (H) Cutting edge of labrum showing small teeth. Scale bars in μm.
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Figure 7. Conchoderma hunteri. (A) Cirrus I, showing two filamentary appendages at base region. (B) Posterior ramus, showing serrulate setae. (C) Anterior ramus, showing serrulate setae. (D) Protuberant segment of cirrus I, showing serrulate setae. (E) Cirrus II. (F) Posterior ramus showing serrulate setae. (G) Anterior ramus, showing serrulate setae. (H) Intermediate segment of anterior ramus, showing serrulate setae. Scale bars in μm.
Figure 7. Conchoderma hunteri. (A) Cirrus I, showing two filamentary appendages at base region. (B) Posterior ramus, showing serrulate setae. (C) Anterior ramus, showing serrulate setae. (D) Protuberant segment of cirrus I, showing serrulate setae. (E) Cirrus II. (F) Posterior ramus showing serrulate setae. (G) Anterior ramus, showing serrulate setae. (H) Intermediate segment of anterior ramus, showing serrulate setae. Scale bars in μm.
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Figure 8. Conchoderma hunteri. (A) Cirrus III, note single filamentary appendage at base. (B) Intermediate segments of posterior ramus, showing serrulate setae. (C) Distal segment of posterior ramus. (D) Magnified segment of anterior ramus of cirrus III, showing serrulate setae. (E) Cirrus IV. (F) Serrulate-type setae at cirrus IV. (G) Distal end of posterior ramus of cirrus IV, showing short robust simple setae. (H) Intermediate segment of cirrus IV, showing serrulate setae. Scale bars in μm.
Figure 8. Conchoderma hunteri. (A) Cirrus III, note single filamentary appendage at base. (B) Intermediate segments of posterior ramus, showing serrulate setae. (C) Distal segment of posterior ramus. (D) Magnified segment of anterior ramus of cirrus III, showing serrulate setae. (E) Cirrus IV. (F) Serrulate-type setae at cirrus IV. (G) Distal end of posterior ramus of cirrus IV, showing short robust simple setae. (H) Intermediate segment of cirrus IV, showing serrulate setae. Scale bars in μm.
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Figure 9. Conchoderma hunteri. (A) Cirrus V. (B) Intermediate segment of cirrus V showing serrulate setae. (C) Distal segment of cirrus V, showing robust simple setae. (D) Intermediate segment of cirrus V showing serrulate setae. (E) Cirrus VI. (F) Serrulate setae at intermediate segment of cirrus VI. (G) Penis. (H) Apex of penis. Scale bars in μm.
Figure 9. Conchoderma hunteri. (A) Cirrus V. (B) Intermediate segment of cirrus V showing serrulate setae. (C) Distal segment of cirrus V, showing robust simple setae. (D) Intermediate segment of cirrus V showing serrulate setae. (E) Cirrus VI. (F) Serrulate setae at intermediate segment of cirrus VI. (G) Penis. (H) Apex of penis. Scale bars in μm.
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Figure 10. Conchoderma hunteri. (A) Maxilla. (B) Serrulate setae at outer margin of maxilla. (C) Serrulate setae at inner margin of maxilla. (D) Maxillule, showing three zig-zag regions at cutting edge. (E) Large spine of maxillule. (F) Simple setae at the base of large spine of maxillule. (G) Cutting edge of maxillule. (H) Serrulate-type setae at cutting edge of maxillule. Scale bars in μm.
Figure 10. Conchoderma hunteri. (A) Maxilla. (B) Serrulate setae at outer margin of maxilla. (C) Serrulate setae at inner margin of maxilla. (D) Maxillule, showing three zig-zag regions at cutting edge. (E) Large spine of maxillule. (F) Simple setae at the base of large spine of maxillule. (G) Cutting edge of maxillule. (H) Serrulate-type setae at cutting edge of maxillule. Scale bars in μm.
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Figure 11. Conchoderma hunteri. (A) Mandible, showing five teeth. (B) First tooth of mandible. (C) 3rd tooth of the mandible. (D) Small 5th tooth of the mandible, size comparable to the inferior angle. (E) Mandibulary palp. (F) Serrulate setae at margin of palp. (G) Labrum. (H) Cutting edge of labrum showing small teeth. Scale bars in μm.
Figure 11. Conchoderma hunteri. (A) Mandible, showing five teeth. (B) First tooth of mandible. (C) 3rd tooth of the mandible. (D) Small 5th tooth of the mandible, size comparable to the inferior angle. (E) Mandibulary palp. (F) Serrulate setae at margin of palp. (G) Labrum. (H) Cutting edge of labrum showing small teeth. Scale bars in μm.
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Figure 12. Comparison of mandible from four different specimens of C. virgatum (A–D) and four different specimens of C. hunteri (E–H). Note consistent differences in the size of 5th tooth (indicated by arrows) between C. virgatum and C. hunteri. Scale bars in μm.
Figure 12. Comparison of mandible from four different specimens of C. virgatum (A–D) and four different specimens of C. hunteri (E–H). Note consistent differences in the size of 5th tooth (indicated by arrows) between C. virgatum and C. hunteri. Scale bars in μm.
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Figure 13. A Maximum likelihood (ML) phylogeny tree reconstructed with COI sequences by using MEGA11. Bootstrap values of neighbor joining (NJ), maximum likelihood (ML), and posterior probability of Bayesian inference (BI) are shown at the nodes when >50%, respectively. The species delimitation results of ASAP, PTP, and GMYC are indicated with vertical bars. The number of ASAP represent 3 models used for analysis, (1) Jukes-Cantor model (JC69), (2) Kimura 2-parameter model (K2P), and (3) Simple distance (p-distances). The number of PTP analysis represent (4) Bayesian Poisson tree processes (bPTP) and (5) the PTP result of the Maximum likelihood solution.
Figure 13. A Maximum likelihood (ML) phylogeny tree reconstructed with COI sequences by using MEGA11. Bootstrap values of neighbor joining (NJ), maximum likelihood (ML), and posterior probability of Bayesian inference (BI) are shown at the nodes when >50%, respectively. The species delimitation results of ASAP, PTP, and GMYC are indicated with vertical bars. The number of ASAP represent 3 models used for analysis, (1) Jukes-Cantor model (JC69), (2) Kimura 2-parameter model (K2P), and (3) Simple distance (p-distances). The number of PTP analysis represent (4) Bayesian Poisson tree processes (bPTP) and (5) the PTP result of the Maximum likelihood solution.
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Table
Table 1. Specimen information and GenBank accession numbers for DNA sequences.
Table 1. Specimen information and GenBank accession numbers for DNA sequences.
SpeciesSpecimen VoucherLocalityGenBank Accession NumbersReference
Lepas anseriferaKU204318Apra Harbor, GuamKU204318[34]
Lep_sp_001Taitung, Taiwan ON938319 Present study
Scalpellum scalpellumKT208441North Sea, TaiwanKT208441[35]
Octolasmis corKC138499Pingtung, TaiwanKC138499[36]
Octolasmis angulataKC138498Ha Long Bay, VietnamKC138498Unpublished
Lepas australisGU993634ChileGU993634Unpublished
Paralepas laxusEU884174-EU884174[37]
Octolasmis orthogoniaEU884173-EU884173
Heteralepas japonicaEU884169-EU884169
Oxynaspis sp.CD_3257_1MadagascarON938312Present study
Conchoderma hunteriCon_h_01Panglao Island, PhilippinesKF484213[38]
Con_h_02KC138463
Con_h_03Penghu, TaiwanON938315
Con_h_04ON938315
Conchoderma virgatumCon_v_02Pingtung, TaiwanKC138464[36]
Con_sp_01Kagoshima, JapanON938317
Conchoderma auritumMT563438-MT563438Unpublished
MT563441-MT563441
KU204353Portland, USAKU204353[34]
Table
Table 2. Kimura 2-parameter (K2P) distances of COI sequences between species using MEGA X. The lower left of the matrix are the mean distances, and the upper right of the matrix are the SD. Bolded values indicate the comparison between C. hunteri and C. virgatum.
Table 2. Kimura 2-parameter (K2P) distances of COI sequences between species using MEGA X. The lower left of the matrix are the mean distances, and the upper right of the matrix are the SD. Bolded values indicate the comparison between C. hunteri and C. virgatum.
123456789101112
1. Conchoderma hunteri 0.0160.0320.0350.0350.0360.0380.0360.0360.0410.0450.044
2. Conchoderma virgatum0.097 0.0300.0350.0420.0340.0360.0360.0380.0420.0410.040
3. Conchoderma auritum0.2570.242 0.0390.0390.0430.0440.0420.0400.0490.0440.046
4. Lepas australis0.2780.2850.300 0.0320.0330.0420.0340.0390.0420.0420.039
5. Lepas anserifera0.2760.3390.3130.257 0.0350.0510.0390.0440.0430.0440.044
6. Heteralepas japonica0.2880.2820.3450.2580.278 0.0310.0360.0350.0370.0410.038
7. Salpellum scalpellum0.3110.2980.3460.3340.3960.255 0.0350.0360.0390.0400.036
8. Paralepas laxus0.2790.2810.3360.2640.3200.2780.278 0.0310.0420.0310.039
9. Oxynaspis sp.0.2780.3060.3200.3070.3530.2770.2810.231 0.0350.0310.035
10. Octolasmis orthogonia0.3210.3360.3940.3370.3460.3070.3150.3500.275 0.0300.033
11. Octolasmis angulata0.3500.3330.3630.3310.3430.3300.3210.2480.2350.236 0.029
12. Octolasmis cor0.3540.3310.3990.3040.3580.3130.2880.3060.2680.2720.206
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Chan, B.K.K.; Chen, Y.-H. Distinguishing Long-Discussed Cryptic Species of the Epibiotic Goose-Neck Barnacle of the Genus Conchoderma (Thoracicalcarea: Lepadidae) with Integrative Taxonomy. Diversity 2022, 14, 593. https://doi.org/10.3390/d14080593

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Chan BKK, Chen Y-H. Distinguishing Long-Discussed Cryptic Species of the Epibiotic Goose-Neck Barnacle of the Genus Conchoderma (Thoracicalcarea: Lepadidae) with Integrative Taxonomy. Diversity. 2022; 14(8):593. https://doi.org/10.3390/d14080593

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Chan, Benny K. K., and Yu-Hsuan Chen. 2022. "Distinguishing Long-Discussed Cryptic Species of the Epibiotic Goose-Neck Barnacle of the Genus Conchoderma (Thoracicalcarea: Lepadidae) with Integrative Taxonomy" Diversity 14, no. 8: 593. https://doi.org/10.3390/d14080593

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