Research Article |
Corresponding author: Mariastella Colomba ( mariastella.colomba@uniurb.it ) Academic editor: Pavel Stoev
© 2022 Mariastella Colomba, Gabriella Lo Verde, Fabio Liberto, Armando Gregorini, Ignazio Sparacio.
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:
Colomba M, Lo Verde G, Liberto F, Gregorini A, Sparacio I (2022) Molecular and biometric data on Carabus (Macrothorax) morbillosus Fabricius, 1792 (Coleoptera, Carabidae) from Mid Mediterranean areas. ZooKeys 1127: 119-134. https://doi.org/10.3897/zookeys.1127.84920
|
The present study was carried out using molecular and biometric data of Carabus (Macrothorax) morbillosus from mid-Mediterranean areas to determine additional information on basal relationships among its representative subspecies. To this aim, two different kinds of approach were employed, including a morphometric analysis of four morphological parameters (i.e., elytra length, elytra width, pronotum length, pronotum width) of 128 specimens, and a Bayesian genetic analysis of 44 cytochrome oxidase subunit I (COI) partial sequences (i.e., 38 examined for the first time and six retrieved from GenBank database). Representative populations of C. (M.) morbillosus were sampled in four countries, namely Italy, Malta, Spain, and Tunisia. The present findings support the validity of four C. (M.) morbillosus subspecies, specifically C. (M.) m. alternans, C. (M.) m. bruttianus, C. (M.) m. constantinus, and C. (M.) m. macilentus, and redefine these subspecies’ distributions. Notably, within the C. (M.) m. constantinus clade, two (i.e., Sardinia/Tuscany and Lampedusa) out of the three subgroups appear as homogeneous geographical groupings.
Bayesian analysis, biogeography, carabids, COI, elytra, morphometric analysis, pronotum, taxonomy
The genus Carabus Linnaeus, 1758 (Coleoptera, Carabidae) includes about 1000 species currently classified in over 91 subgenera. This genus is widespread in the Holarctic area but nearly all species are distributed in the Palearctic region including Japan, Iceland, Canary Islands, and North Africa, with only a few (11 species) in North America (
Carabids are mostly nocturnal predators represented by numerous brachypterous (i.e., wingless) species with low dispersal power, living in restricted areas, sometimes punctiform, and with extreme specialization towards particular environments (forests, grasslands, or agricultural landscapes) and prey (snails, earthworms, or caterpillars). Such a high degree of ecological differentiation is represented by numerous (morphological) subspecific forms (
Molecular genetic studies confirmed a substantial monophyly of the morphological subgroups of Carabus (
Within the large Carabidae family, speciation processes are probably due to geological and paleo-ecological events, and, for the Euro-Mediterranean area, they can be explained by the Eurasian forest fragmentation consequent to the Miocene climatic changes and subsequent Plio-Pleistocene climatic events (see also
The subgenus Macrothorax was described by
Currently (see
C. (M.) morbillosus Fabricius, 1792 (S-France, S-Spain, N-Morocco, N-Algeria, N-Tunisia, Corse, Sardinia, Tyrrhenian central Italy, Southern Calabria, Sicily, Sicilian islands and Malta);
C. (M.) rugosus Fabricius, 1792 (S-Spain, Portugal, N-Morocco);
C. (M.) aumontii Lucas, 1849 (NE-Morocco, NW-Algeria), type species;
C. (M.) planatus Chaudoir, 1843 (Sicily);
C. (M.) meurguesianus Ledoux, 1990 (Morocco) (Fig.
These species live in varied habitats. Carabus (M.) morbillosus and C. (M.) aumontii mostly occur at low and medium altitudes in dense Mediterranean shrubland or palm forest soils with sufficient vegetation coverage, but also in areas with sparse vegetation or stony ground. Carabus (M.) rugosus and C. (M.) planatus are present in mountain and woodland environments.
With the exception of C. (M.) planatus, subspecies limited to well-defined geographic areas are ascribed to each of these species.
Considering only C. (M.) morbillosus, these subspecies include:
Carabus (M.) m. morbillosus Fabricius, 1792, locus typicus: “Mauretania” (
Carabus (M.) m. alternans Palliardi, 1825, locus typicus: Sicilia (
Carabus (M.) m. macilentus Lapouge, 1899, “sud de l’Espagne” (Lapouge 1899).
Carabus (M.) m. cychrisans Lapouge, 1899, NW-Algeria: Oran env. ? Maghnia (Lapouge 1899).
Carabus (M.) m. galloprovincialis Lapouge, 1910, locus typicus: Le Muy, Var, France (Lapouge 1910;
Carabus (M.) m. constantinus Lapouge, 1899, locus typicus: “Constantine [Algeria]” (Lapouge 1899).
Carabus (M.) m. bruttianus Born, 1906, locus typicus: “Calabria” (
Carabus (M.) m. arborensis Krausse, 1908, locus typicus: “Asuni [Sardinia, Italy]” (
Carabus (M.) m. corsicanus Lapouge, 1913, locus typicus: “Corsica” (Lapouge 1913).
Carabus (M.) m. lampedusae Born, 1925, locus typicus: “Lampedusa” (
Carabus (M.) m. cheminorum Deuve, 1988.
Many of these taxa are not accepted or have had different interpretations (
The taxonomy and morphological characteristics of the main morbillosus subspecies accepted so far are briefly reported below.
Carabus (M.) m. morbillosus: N-Algeria SE-France (but the French populations were probably introduced from Kabylia: subsp. cheminorum). Pronotum widened anteriorly, sides arcuate, slightly narrowed anteriorly side, elytra elongate-ovate, subconvex, elytral sculpture with primary intervals catenulate, convex, 4th secondary interval evident, complete, tertiary intervals reduced, broken in rows of granulations, dorsal surface often darker and more polychromous, in some populations green to blue-violet.
Carabus (M.) m. macilentus: SE-Spain (Murcia, Catalonia), Algeciras (Cádiz), Balearic Islands. Pronotum narrowed anteriorly, the sides slightly arched, very short tertiary intervals, depressed elytra, cupreous dorsal surface normally dark, or greenish, the disc of the pronotum often blackish.
Carabus (M.) m. constantinus: NE-Algeria, Tunisia, Italy (Tuscany, Lazio: probably introduced), Sardinia, Corse, Lampedusa, SE-France (introduced probably from Corse). Elytra more convex; intervals less convex, 4th secondary interval fully reduced, tertiary intervals granulated but more evident than in the typical form, dorsal surface more constantly metallic bronze to reddish-cupreous.
Carabus (M.) m. alternans: Sicily, Calabria (Aspromonte), Basilicata, Malta. This population is differentiated from the other populations by a large shiny pronotum flattened posteriorly, with maximum width at middle and constricted forward; primary intervals elongated and slightly salient, secondary ribs depressed, tertiary intervals less raised than secondary ones, 1st elytral interstria deeply punctured with points sometimes juxtaposed; apex of aedeagus relatively short and wide, elytra elongate, rounded and dilated in the rear third, elytra apex short and sightly sinuate at sides.
A few years ago,
Carabus (M.) m. lampedusae is similar to C. (M.) m. constantinus but shows a large and convex body shape and is less bright in color. Dark pronotum with basal sulci large and deep, side sinuate before hind angle, primary intervals wider, 1st elytral interstria with points on the surface, well separated from each other.
Carabus (M.) m. bruttianus is similar to C. (M.) m. alternans but is smaller and convex on elytron apex, less shiny, pronotum narrower and slightly rounded forward with maximum width in the fore half, elytra evidently shorter and oval, primary intervals in granules shorter and less raised, elytron apex stretched and clearly sinuate at side.
Likewise,
To date interpretation of C. (M.) morbillosus subspecies remains elusive. To contribute to this problem, we used both a morphometric analysis of four morphological characters (i.e., elytra length, elytra width, pronotum length, and pronotum width), and a genetic analysis of a fragment of the cytochrome oxidase subunit I (COI) gene to determine additional information on the basal relationships among representative populations of C. (M.) morbillosus in mid-Mediterranean areas. Our focus was on populations inhabiting central mainland Italy, Sardinia, Sicily, circum-Sicilian islands, Malta, Spain, the Balearic Islands, and Tunisia.
A total of 128 Carabus (M.) morbillosus male specimens were studied in the morphometric analysis. Samples were collected in Italy (Lampedusa, Sardinia, Calabria, Sicily, including four locations throughout the island, plus Messina province, which is interesting for its biogeographical connections with Calabria), Tunisia, and the Balearic Islands.
For each specimen four characters were measured: length of elytra (EL), width of elytra (EW), length of pronotum (PL), width of pronotum (PW).
Morphometric characters were used in an exploratory cluster analysis (complete linkage, Euclidean distance) to determine if the combinations of biometric characters allow to delimit groups concordant with the subspecies. Afterwards, a discriminant analysis was performed to assess the usefulness of the recorded variables to identify groups. A principal component (PC) analysis was then performed using the same four morphometric factors. Since one character (PL) was not available for one specimen from Sardinia, 127 male specimens were used for the analyses. Moreover, analysis of mean differences of morphometric characters among groups was then performed with ANOVA, after data normalization by means of a Box-Cox transformation. All analyses were concluded with Tukey post hoc tests to compare the groups for each character (p < 0.05). Minitab software has been used throughout for all statistical analyses.
Molecular analysis was performed on 38 specimens of C. (M.) morbillosus from several localities: Malta [(collection site not available, (CMAL)]; Vizzini [Italy: Sicily (VIZ)]; Custonaci [Italy: Sicily (CUST)]; Corleone, Ficuzza [Italy: Sicily (FIC)]; Messina [Italy: Sicily (MES); Reggio Calabria [Italy: S-Calabria (RCAL)]; Lipari [Italy: Aeolian Islands (LIP)]; Olmedo Prepalzos [Italy: N-Sardinia (OLM)]; Capoterra and Is Cannoneris [Italy: S-Sardinia (SARD)]; Follonica [Italy: Tuscany (FOL)]; Castiglione della Pescaia [Italy: Tuscany (CAST)]; Lampedusa [Italy: Sicily, Pelagie Islands, (LAMP)]; Cap Gammarth (Tunisia (CGAM)]; Ses Mongetes, Citadella de Menorca [Spain: Balearic Islands, Menorca (MEN)]; and Lloc de Monges, [Spain: Balearic Islands, Menorca (MON)] (Fig.
Samples were stored at −20 °C in test tubes. Total genomic DNA was isolated from a small piece of tissue taken from the ethanol-preserved specimens. The extractions were carried out using the Wizard Genomic DNA Purification Kit (Promega). All the DNA extractions were kept at 4 °C for short-time use. Undiluted or different dilutions (1:10–1:50, based on the DNA concentration) of each DNA extraction were used as templates for PCR amplification of a portion of the cytochrome oxidase subunit I (mt-COI) gene.
COI amplicons were obtained by the universal internal primers LCO1490 and HCO2198 as in
All sequences generated in the present study were deposited in NCBI GenBank (OM681023–OM681060).
Phylogenetic analyses were conducted in BEAST 1.6.1 (
In addition, homologous sequences (retrieved from GenBank) of Carabus rugosus (JQ689882, JQ689892), C. morbillosus alternans (JQ646591), C. morbillosus (JQ689896-JQ689898, JQ689883, JX279622), C. planatus (JQ646589), Calosoma sycophanta (JQ693413), and Cychrus semigranosus (JQ689876) were included. Campalita auropunctatum (JQ689899) was used as outgroup (OG).
The dendrogram obtained from the cluster analysis (Fig.
The results of the discriminant analysis, conducted on the groups from Sicily, Sardinia, Tunisia, Lampedusa, Messina/Calabria, and Spain/Balearic Islands, reveals that the proportion of correct attribution is 0.646 (Table
Classification of Carabus (Macrothorax) morbillosus male specimens of the different groups determined by the discriminant analysis performed on all parameters. Coefficients of the discriminant function show the impact of each parameter in the correct attribution to the four groups.
Lampedusa | Messina/Calabria | Sardinia | Sicily | Spain/Balearic Is. | Tunisia | |
---|---|---|---|---|---|---|
Lampedusa | 14 | 0 | 3 | 1 | 0 | 4 |
Messina/Calabria | 0 | 28 | 2 | 4 | 3 | 0 |
Sardinia | 1 | 1 | 8 | 2 | 0 | 1 |
Sicily | 1 | 3 | 5 | 11 | 0 | 0 |
Spain/Balearic Is. | 2 | 1 | 2 | 2 | 14 | 1 |
Tunisia | 5 | 0 | 0 | 0 | 1 | 7 |
Total N | 23 | 33 | 20 | 20 | 18 | 13 |
N correct | 14 | 28 | 8 | 11 | 14 | 7 |
Proportion | 0.61 | 0.85 | 0.4 | 0.55 | 0.78 | 0.54 |
N = 127 | N correct = 82 | Proportion correct = 0.646 | ||||
Linear Discriminant Function for groups | ||||||
Lampedusa | Messina/Calabria | Sardinia | Sicily | Spain/Balearic Is. | Tunisia | |
Constant | −345.05 | −274.38 | −298.79 | −303.67 | −332.05 | −366.62 |
Elytra width | −0.20 | −0.16 | −0.11 | −0.08 | −0.21 | −0.20 |
Elytra length | 2.20 | 1.73 | 1.84 | 1.86 | 2.01 | 2.27 |
Pronotum width | 1.91 | 1.57 | 1.88 | 1.63 | 1.64 | 1.78 |
Pronotum length | 2.24 | 2.84 | 2.43 | 2.71 | 2.97 | 2.56 |
Results of the statistical analysis for the examined morphometric parameters are reported in Table
Biometrics (mean ± S.E.) of males of the different Carabus (Macrothorax) morbillosus groups. Different letters within the column indicate significant differences among group means (One-way ANOVA performed after Box-Cox transformation of data: EW F5,122 = 11.52; EL F5,122 = 46.58; PW F5,122 = 22.94; PL F5,121 = 10.84 followed by Tukey post-hoc test, p < 0.05).
Groups | No. | EW ± SE (min-max) | EL ± SE (min-max) | PW ± SE (min-max) | PL ± SE (min-max) |
---|---|---|---|---|---|
Calabria/Messina | 33 | 103.94 ± 2.36 c | 166.45 ± 1.14 d | 71.55 ± 0.61 d | 58.36 ± 0.40 b |
(86–150) | (151–178) | (65–81) | (55–63) | ||
Spain/Balearic Is. | 18 | 108.28 ± 0.94 bc | 184.72 ± 1.79 b | 77.72 ± 1–16 bc | 63.33 ± 0.63 a |
(100–114) | (170–200) | (70–88) | (59–68) | ||
Tunisia | 13 | 116.62 ± 1.99 ab | 197.31 ± 2.97 a | 82.38 ± 1.06 a | 63.85 ± 0.96 a |
(105–130) | (180–220) | (77–90) | (59–70) | ||
Lampedusa | 23 | 113.43 ± 1.51 ab | 191.43 ± 1.70 ab | 81.61 ± 1.09 ab | 60.65 ± 0.96 b |
(100–133) | (180–210) | (74–90) | (54–70) | ||
Sardinia | 21 (20 for PL) | 124.71 ± 5.28 ab | 175.57 ± 1.79 c | 77.60 ± 0.70 c | 58.57 ± 0.72 b |
(99–170) | (153–190) | (70–82) | (53–64) | ||
Sicily | 20 | 131.00 ± 5.48 a | 177.25 ± 1.44 c | 76.15 ± 0.85 c | 60.45 ± 0.34 ab |
(98–180) | (166–192) | (70–82) | (58–64) |
The PC analysis indicates that the four morphometric characters explained 83.2% of all variance, mainly related to PL, PW, and EL (PC1, 61.6%) (Fig.
As shown in Fig.
50% majority rule Bayesian tree inferred from dataset including partial sequences of the mitochondrial COI genes available in the present paper along with homologous sequences retrieved from GenBank (see text for details). Nucleotide substitution model: HKY + G (gamma = 0.128). Numbers above branches represent Bayesian posterior probabilities. Scale bar represents units of length in expected substitutions per site.
The results of the morphometric and molecular analyses in this study show a significant agreement between hypothesized relationships of taxa. Combining information from the similarity tree and the phylogenetic tree, the validity of the subspecies alternans, bruttianus, constantinus, and macilentus is supported.
The subspecies bruttianus is only separated by a small genetic distance (ca 1%) from alternans, but the subspecific rank is supported by the tree topology. In addition, a comparison between the Calabria/Messina clustering obtained with morphometric analysis and the MES+RCAL+LIP cluster in the Bayesian tree clearly supports the validity of the subspecies bruttianus, as proposed by
Within the large clade constantinus, out of three subgroups, two homogeneous geographic groupings were found in Sardinia (including also Tuscany) and Lampedusa, whereas the third one (Tunisia) appears to be more heterogeneous. Of the Sardinian specimens, all individuals cluster within the constantinus group, while the morphometric analysis shows them to be closer to alternans.
The molecular similarity between Sardinian and Tuscan populations is in agreement with their morphological similarity which, depending on different hypotheses, is considered the result of ancient passive transport (i.e., by anthropogenic transport, perhaps by the Phoenicians;
Although Lampedusa specimens are all included in a homogeneous geographical subgroup, the subspecific rank is only partially supported by the tree topology using COI data. However, such an outcome does not necessarily affect the validity of the subspecies which was diagnosed morphologically. Combining molecular with morphological monophylies, a subgenus is supported, but more in-depth study is needed by analyzing more morphological characters, more beetle specimens, and more genes (at least one nuclear) to obtain a clearer insight on the evolutionary paths followed by morbillosus in Italy and Tunisia. Of course, this larger study must also include beetles from Algeria, northern Morocco, and southeastern Spain.
In conclusion, our results provide new evidence supporting the validity of the C. (M.) morbillosus subspecies, C. (M.) m. alternans, C. (M.) m. bruttianus, C. (M.) m. constantinus, and C. (M.) m. macilentus, and we can redefine their distribution in mid-Mediterranean areas.
One latter consideration refers to C. (M.) planatus which was shown in the phylogenetic tree as the most distant Macrothorax species analysed. It is an endemic species that lives exclusively in the Nebrodi and Madonie woods of Sicily (
We are grateful to all friends and colleagues who provided help and specimens used in the study. They are, in alphabetical order, Marcello Arnone, Marco Bastianini, Michele Bellavista, Giuseppe Maraventano, David Mifsud, Pietro Lo Cascio, Stefano Nappini, Roberto Pantaleoni, Elena Prazzi, Josep Quintana Cardona, Ivan Rapuzzi, and Marcello Romano. We also wish to thank José Serrano Marino for his comments and suggestions on the manuscript and Robert Forsyth for English revision.