Research Article |
Corresponding author: Ronald Vonk ( ronald.vonk@naturalis.nl ) Academic editor: Charles Oliver Coleman
© 2017 Domenico Davolos, Elvira De Matthaeis, Leonardo Latella, Ronald Vonk.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Davolos D, De Matthaeis E, Latella L, Vonk R (2017) Cryptorchestia ruffoi sp. n. from the island of Rhodes (Greece), revealed by morphological and phylogenetic analysis (Crustacea, Amphipoda, Talitridae). ZooKeys 652: 37-54. https://doi.org/10.3897/zookeys.652.11252
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A new Cryptorchestia species, Cryptorchestia ruffoi Latella & Vonk, sp. n. from the island of Rhodes in south-eastern Greece, can be distinguished on the basis of morphological and phylogenetic data. Morphological analysis and DNA sequencing of mitochondrial and nuclear protein-coding genes indicated that this species is related to C. cavimana (Cyprus) and C. garbinii (Mediterranean regions, with a recent northward expansion). Results supported a genetic separation between the Cryptorchestia species of the east Mediterranean regions and those of the northeast Atlantic volcanic islands examined in this study (C. canariensis, C. gomeri, C. guancha, and C. stocki from the Canary islands, C. monticola from Madeira, and C. chevreuxi from the Azores). The Mediterranean and Atlantic Cryptorchestia species appear to be also morphologically distinct. Cryptorchestia ruffoi sp. n., C. cavimana, C. garbinii, and C. kosswigi (Turkish coast) clearly have a small lobe on the male gnathopod 1 merus. This character was the main diagnostic difference between Cryptorchestia (sensu Lowry, 2013) and Orchestia. However, among the six northeast Atlantic island Cryptorchestia species only C. stocki has a small lobe on the merus of gnathopod 1. Reduction or loss of the lobe in the Atlantic Island species cannot be ruled out; however, molecular phylogenetic analysis leads us to presume that this lobe independently evolved between the east Mediterranean Cryptorchestia species and C. stocki from Gran Canaria.
biogeography, freshwater, Greek islands, molecular phylogeny, taxonomy
The genus Cryptorchestia Lowry & Fanini, 2013 is partitioned from Orchestia Leach, 1814, and is associated with freshwater-soaked leaf litter (
Here Cryptorchestia ruffoi sp. n. is described from the island of Rhodes, Greece. Specimens were first collected during the military occupation of Rhodes by the Italian army in 1928: the geologist Angelo Pasa of the Museo Civico di Storia Naturale of Verona found two talitrid specimens in a spring on Monte Smith (Fig.
A morphological characterisation forms part of this study. In order to gain insight into its position in relation to other Cryptorchestia species of the Mediterranean and North East Atlantic areas, a phylogenetic analysis was performed on DNA sequences of both mitochondrial (mt) and nuclear gene fragments, cytochrome oxidase I (COI), and histone H3 (H3), respectively. In particular, focus was on Cryptorchestia ruffoi sp. n. from Rhodes, C. cavimana (Heller, 1865) from Cyprus, C. garbinii Ruffo, Tarocco and Latella, 2014, from mainland Europe, C. canariensis (Dahl, 1950), C. gomeri (Stock, 1989), C. guancha (Stock & Boxshall, 1989), and C. stocki (Ruffo, 1990) from the Canary Islands, C. monticola (Stock & Abreu, 1992) from Madeira, and C. chevreuxi (De Guerne, 1887) from Terceira, Azores.
Morphology. The specimens analysed were collected in two localities from Rhodes (Fig.
PCR amplification and DNA sequencing. Specimens of Cryptorchestia ruffoi sp. n. from Rhodes, C. cavimana from Cyprus, C. garbinii from Europe and Macedonia, C. canariensis, C. gomeri, C. guancha, and C. stocki from the Canary Islands, C. monticola from Madeira, and C. chevreuxi from Terceira, Azores were stored in ethanol (Table
Mediterranean and North-East Atlantic Cryptorchestia species employed in the molecular analysis. Shown are the mitochondrial COI gene region (363 bp), the H3 histone gene fragment (330 bp), the sampling locations, and the GenBank accession number (acc. no.). Platorchestia platensis, used in this study as outgroup species, is also reported. NA = not available.
Species | Sampling locality | acc. no. | ||
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COI | H3 | Reference | ||
Cryptorchestia canariensis (Dahl, 1950) | Gran Canaria, Canary Islands, Spain | KY225807 | KY225817 | present study |
Cryptorchestia cavimana (Heller, 1865) | Troodos Mountains, Cyprus | KY225808 | KY225818 | present study |
Cryptorchestia chevreuxi (de Guerne, 1887) (ZMA.CRUS.A.108.587; Leiden Museum) | Terceira, Azores, Portugal | NA | KY225819 | present study |
Cryptorchestia garbinii Ruffo, Tarocco & Latella 2014 | Lake Ohrid, Macedonia | KY225809 | KY225820 | present study |
Dijon, France | KY225810 | KY225821 | present study | |
Latium, Italy | KY225811 | KY225822 | present study | |
Cryptorchestia guancha (Stock & Boxshall, 1989) | Zapata, Tenerife, Canary Islands, Spain | KY225812 | KY225823 | present study |
Cryptorchestia gomeri (Stock, 1989) | La Gomera, Canary Islands, Spain | NA | AM748658 |
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Cryptorchestia monticola (Stock & Abreu 1992) (paratype; Leiden Museum) | Madeira Island, Portugal | KY225813 | KY225824 | present study |
Cryptorchestia ruffoi Latella & Vonk, sp. n. | Rhodes Island, Greece | KY225814 | KY225825 | present study |
Cryptorchestia stocki (Ruffo, 1990) (paratype; Museo Civico di Storia Naturale, Verona) | Gran Canaria, Canary Islands, Spain | KY225815 | KY225826 | present study |
Platorchestia platensis (Krøyer, 1845) | Capri Island, Italy | KY225816 | KY225827 | present study |
Genomic DNA was extracted from pereopods or whole organisms using QIAamp DNA Mini kit (QIAGEN). A PCR product of ca. 400 base pairs (bp) was amplified from the gene encoding the mt COI (some of our samples were old museum specimens in which mitochondrial DNA was degraded and consequently produced only short DNA sequences). The PCR-mediated reaction was performed using the primers BI-COI and SUBIR cited in
Bioinformatic analysis. The nucleotides obtained in this study and the amino acid residues inferred were compared with sequence data accessible in the GenBank databases at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) using the BLASTN algorithm. The nucleotide sequence alignments were made in ClustalX (1.8) using the default parameters. Evolutionary analyses for the combined mt COI and nuclear histone H3 gene sequences were conducted in MEGA7 (
Island of Rhodes, Greece. A spring on Monte Smith and in streams flowing out of the springs called Epta Pyges (Seven Springs), Municipality of Archangelos.
Species named after Sandro Ruffo (1915 – 2010), tutor to us all, who worked on Mediterranean talitrid amphipods from an early stage.
Type specimens. Holotype male (15.7 mm): Greece, Rhodes Island, Rhodes, Monte Smith, near a little spring, June 1928, (labelled: Rodi VI-928 Monte Smiti, vicino piccola sorgente) (MSNVRCr 589). Paratypes: 9 males, 25 females; Greece, Rhodes, Psintos, Epta Piges spring's stream, 36°15'10.9'N - 28°06'49.3'E, 7/8-VII-2010, A. & L. Latella, V. Lencioni leg.: 1 male (vial
Cryptorchestia garbinii Ruffo, Tarocco & Latella, 2014 : Italy, Lombardy, Brescia province, Lake Garda, between Desenzano and Padenghe, 45°29'N–10°30'E, V-1895, A. Garbini leg.; Venetia, Verona province, Lake Garda, Peschiera, 45°26'51"N–10°41'39"E, 18-IV-2010 L. Latella, V. Lencioni leg.
Cryptorchestia cavimana (Heller, 1865): Cyprus, Troodos Mountains, Kaledonia falls, 1250 m a.s.l., 9-VI-2000 M. Tarocco leg.; Cyprus, Troodos Mountains, between Prodromos and Troditissa, 1300 m a.s.l., 10-VI-2000, M. Tarocco leg.
Gnathopd 2 propodus sinusoid palmar margin with its strongest incursion close to the anterior side. Maxilla 1 with vestigial palp present on the outer lobe. Pereopod 5 with four groups of setae on the posterior margin in females and five groups in the males. Antenna first flagellum segment sometimes fused with second, forming a longer unit.
Based on adult males with an average length of 12.20 mm (Table
Cryptorchestia ruffoi sp. n. Measurements of body length and antennae, showing differences in male and female individuals.
Total body length | Length of antenna 1 | Length of antenna 2 | |
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♂ | 11.60 | 1.37 | 4.75 |
♂ Holotype | 15.70 | 1.64 | 5.99 |
♂ | 14.17 | 1.91 | 7.12 |
♂ | 10.77 | 1.29 | 4.11 |
♂ | 10.84 | 1.36 | 4.45 |
♂ | 10.14 | 1.21 | 3.80 |
Mean | 12.20 | 1.46 | 5.04 |
♀ | 11.55 | 1.20 | 3.88 |
♀ | 12.42 | 1.54 | 4.75 |
♀ | 10.68 | 1.15 | 3.68 |
♀ | 11.00 | 1.00 | 3.89 |
♀ | 10.11 | 1.01 | 3.90 |
♀ | 11.98 | 1.16 | 4.20 |
♀ | 11.75 | 1.17 | 4.49 |
♀ | 10.38 | 1.00 | 4.00 |
♀ | 11.58 | 0.90 | 4.11 |
♀ | 10.95 | 1.04 | 3.68 |
Mean | 11.24 | 1.12 | 4.06 |
Coxae. Coxal plate 1 with numerous robust setae on distal margin. Coxal plates 2–4 wider than deep, plate 5 elongated, bilobate, plates 6 and 7 smaller.
Pereon. Gnathopod 1 male (Fig.
Pleon. Epimeral plate 2 with a small posteroventral tooth and almost smooth posterior margin. Pleopods 1–3 (Fig.
Female. Based on adult females with an average length of 11,24 mm (Table
The specimens from Rhodes differ in three main characters in comparison to C. cavimana (Cyprus), C. kosswigi (Ruffo, 1949) (Turkish coast), and C. garbinii (Garda Lake), in that they have the sinusoid palmar margin form in the propodus of gnathopod 2 of the male with the strongest incursion closer to the anterior side (Fig.
A Cryptorchestia ruffoi sp. n., gnathopod 2 male, 11.1 mm total body length B Cryptorchestia garbinii from Lake Garda, gnathopod 2 male, 18 mm total body length C Cryptorchestia cavimana from Cyprus, gnathopod 2 male, 14. 8 mm total body length, scale bar 1 mm. D Cryptorchestia ruffoi sp. n. pereopod 7 male E Cryptorchestia garbinii from Lake Garda, pereopod 7 male F Cryptorchestia cavimana from Cyprus, gnathopod 2 male, scale bar 1 mm.
Overall, the morphological differences are subtle and perhaps only have meaning in the combination with a unique genetic signature in its COI and H3 gene fragments.
The major objective of this molecular study, based on DNA sequences of the mt COI and nuclear histone H3 gene regions, was to estimate the evolutionary relationships of Cryptorchestia ruffoi sp. n. in relation to other Cryptorchestia species of the Mediterranean and North East Atlantic areas. DNA sequences from the mt region between the COI and COII genes were also analysed; all the species analysed here showed the peculiar rearrangement (data not shown), originally reported in
Molecular phylogeny by Maximum Likelihood method obtained in a combined analysis using mitochondrial cytochrome oxidase I (COI) gene region (363 bp), and H3 histone (H3) gene fragment (330 bp) sequences (a total of 693 positions in the final dataset) from Cryptorchestia ruffoi sp. n. and other Cryptorchestia species reported in Table
Our results support the proposal by
We thank C. Fišer, A. Latella, V. Lencioni, G. Luquet, P. Oromì, and M. Tarocco for their contribution in collecting some of the samples. We would also like to thank K. van Dorp (Naturalis Biodiversity Center, Leiden, The Netherlands) for her assistance in recovering museum samples of C. chevreuxi and C. monticola; R. Salmaso (Museo Civico di Storia Naturale, Verona, Italy) for her help in assembling the pictures and Nicola Angeli (Museo delle Scienze, Trento, Italy) for the SEM photographs. University students M. Severijns, J. Westerveld (Univ. Leiden), and J. Brugman (Univ. of Amsterdam) are thanked for their useful work in participating on a B.Sc. project of characterstate scores of the new species. The costs for the molecular analysis of this work were supported by Sapienza University of Rome, Rome, Italy. We thank L. Hughes and J. Lowry (Australian Museum, Sydney) for their constructive review comments.