Quantitative morphology and mtDNA reveal that Lasius maltaeus is not endemic to the Maltese Islands (Hymenoptera, Formicidae)

Abstract

Lasius maltaeus Seifert, 2020 was recently described as a Maltese endemic ant based on quantitative morphology, after decades of uncertainties over the identity of the local population, which has a phenotype resembling L. emarginatus (Olivier, 1791). At the same time, Sicilian L. emarginatus populations were discovered to diverge in their mitochondrial DNA to a degree that suggested heterospecificity. Considering the biogeographic similarity of Malta and Sicily, with land bridges connecting them repeatedly until the last glacial maximum, we questioned the assumption that L. maltaeus was endemic to Malta. We integrated quantitative morphology and mtDNA in the study of the Maltese and southern Italian populations phenotypically close to L. emarginatus. We discovered that the range of L. maltaeus extends over most of Sicily, while the true L. emarginatus replace it in the northeastern sector of the island, the nearby Aeolian Islands, and the Italian peninsula. The distributions of L. emarginatus and L. maltaeus in Sicily follow biogeographic patterns recalling the island's complex paleogeographic history. Further investigations should verify the existence of truly Maltese endemic ants, since the status of other allegedly endemic species is not strongly supported.

Full text

Quantitative morphology and mtDNA reveal
that Lasius maltaeus is not endemic to the
Maltese Islands (Hymenoptera, Formicidae)
Mattia Menchetti1*, Enrico Schifani2*, Antonio Alicata3, Roger Vila1
1Institut de Biologia Evolutiva (CSIC-Univ. Pompeu Fabra), Passeig Marítim de la Barceloneta 37-49, 08003
Barcelona, Spain 2Department of Chemistry, Life Sciences and Environmental Sustainability, University
of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy 3Department of Biological, Geological and
Environmental Sciences (DBGES), University of Catania, Via Androne 81, I-95124 Catania, Italy
Corresponding author: Mattia Menchetti (mattiamen@gmail.com; mattia.menchetti@ibe.upf-csic.es)
Academic editor: Francisco Hita Garcia|Received 15 October 2022|Accepted 5 December 2022|Published 17 February 2023
https://zoobank.org/9A24CFED-556E-422D-9570-389B4A5315CA
Citation: Menchetti M, Schifani E, Alicata A, Vila R (2023) Quantitative morphology and mtDNA reveal that
Lasius maltaeus is not endemic to the Maltese Islands (Hymenoptera, Formicidae). Journal of Hymenoptera Research
95: 129–142. https://doi.org/10.3897/jhr.95.96365
Abstract
Lasius maltaeus Seifert, 2020 was recently described as a Maltese endemic ant based on quantitative
morphology, after decades of uncertainties over the identity of the local population, which has a phenotype
resembling L. emarginatus (Olivier, 1791). At the same time, Sicilian L. emarginatus populations were
discovered to diverge in their mitochondrial DNA to a degree that suggested heterospecicity. Considering
the biogeographic similarity of Malta and Sicily, with land bridges connecting them repeatedly until
the last glacial maximum, we questioned the assumption that L. maltaeus was endemic to Malta. We
integrated quantitative morphology and mtDNA in the study of the Maltese and southern Italian
populations phenotypically close to L. emarginatus. We discovered that the range of L. maltaeus extends
over most of Sicily, while the true L. emarginatus replace it in the north-eastern sector of the island, the
nearby Aeolian Islands, and the Italian peninsula. e distributions of L. emarginatus and L. maltaeus
in Sicily follow biogeographic patterns recalling the island’s complex paleogeographic history. Further
investigations should verify the existence of truly Maltese endemic ants, since the status of other allegedly
endemic species is not strongly supported.
Keywords
ants, biogeography, Formicinae, Mediterranean islands, Sicily
* ese authors contributed equally to this study.
JHR 95: 129–142 (2023)
doi: 10.3897/jhr.95.96365
https://jhr.pensoft.net
Copyright Mattia Menchetti et al. 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.
RESEARCH ARTICLE

Mattia Menchetti et al. / Journal of Hymenoptera Research 95: 129–142 (2023)
130
Introduction
e ant genus Lasius Fabricius, 1804 is one of the most ecologically important ant
genera in the Holarctic realm (Hölldobler and Wilson 1990; Seifert 2018, 2020). It
currently counts 125 extant species (Bolton 2022), which belong to two major clades
and at least 10 species groups, many of which independently evolved social parasitism
(Maruyama et al. 2008; Boudinot et al. 2022).
e taxonomy of Lasius ants is considered among the most challenging among
Holarctic ants (Seifert 2018). In particular, the former subgenus Lasius s. str., which
is now known to represent two distinct lineages (the brunneus group and the niger
group, see Boudinot et al. 2022), has witnessed signicant taxonomic changes. It was
once thought to count only seven species (Wilson 1955), but since then this number
increased several times, especially in the last three decades (Van Loon et al. 1990;
Seifert 1991; Seifert 1992, 2020; Schlick-Steiner et al. 2003; Borowiec and Salata
2013; Seifert and Galkowski 2016; Salata and Borowiec 2018; Schär et al. 2022).
e West-Palearctic Lasius emarginatus (Olivier, 1791) is an iconic European spe-
cies characterized by a large geographic range and remarkable bicoloured appearance
(Seifert 2018). However, at least four additional cryptic species with a supercially
similar appearance were revealed during recent years (Seifert 2018, 2020): L. tebessae
Seifert, 1992 from the Maghreb (Seifert 1992, 2020); L. illyricus Zimmermann,
1935 ranging from the Balkans to the Caspian sea, in a large area sympatric with
L. emarginatus (Borowiec and Salata 2013; Schifani and Massa 2020; Seifert 2020);
the Iranian L. persicus Seifert, 2020, partly sympatric with L. illyricus (Seifert 2020);
and L. maltaeus Seifert, 2020 described from Malta.
e recent description of L. maltaeus came after a long debate on the identity of
this Maltese morphotype. Baroni Urbani (1968) attributed the local bicolored Lasius
ants with hairy scapes to L. emarginatus. However, Schembri and Collingwood (1981)
considered the workers’ head pilosity to be too dense, their frontal triangle too shiny
and the scapi of males to be too hairy compared to samples of L. emarginatus from
continental Europe, and instead proposed to classify the Maltese population as a red-
dish phenotype of L. niger (Linnaeus, 1758). Later on, the same authors changed
their opinion, dening the Maltese ants as representatives of a morphospecies dierent
from both L. niger and L. emarginatus (Schembri and Collingwood 1995). e issue
remained uninvestigated until Seifert (2020) included Maltese specimens in a large
West-Palearctic taxonomic revision based on quantitative morphology. He demon-
strated their distinctiveness from both L. niger and L. emarginatus, and described the
morphospecies as L. maltaeus, included within the L. emarginatus complex, empha-
sizing dierences in head pilosity in agreement to the comments by Schembri and
Collingwood (1981).
At the same time, an inventory of the Sicilian ant fauna highlighted a remarkable
genetic distance for the mitochondrial COI of L. emarginatus samples from Sicily com-
pared to those of the nearby Aeolian Islands and of peninsular Italy (Schär et al. 2020).

Lasius maltaeus is not endemic to the Maltese Islands 131
Similar results were also published from Corsica, yet the local population resembling
L. emarginatus was attributed to L. grandis Forel, 1909 based on morphology (Blatrix
et al. 2020; Seifert 2020). Sicily and the Maltese Islands are well-known for their strong
biogeographic similarity (e.g., ake 1985; Fattorini 2011; Salvi et al. 2014; Médail
2022), and were connected repeatedly by land bridges until the last glacial maximum
(Foglini et al. 2016) (light blue line Fig. 1). ese observations led us to investigate the
relationships between the Sicilian populations historically attributed to L. emarginatus
and L. maltaeus, questioning the endemic status of the latter.
Materials and methods
Ant specimens were manually collected and stored in 70–96% ethanol (Suppl. material
1: table S1, Fig. 1). Vouchers are deposited in the authors’ personal collections (see
Suppl. material 1: table S1):
MMBS M. Menchetti pers. coll., Barcelona, Spain;
ESPI E. Schifani pers. coll., Palermo, Italy;
AACI A. Alicata pers. coll., Catania, Italy;
BDEL R. Vila, Buttery Diversity and Evolution Lab coll.
Morphological analysis
e morphological study was performed using stereoscopic microscopes at 45–80×
magnication, in addition to photography-based morphometry. Species were identi-
ed using the keys provided by Seifert (2020).
Morphological measurements were obtained by taking pictures with a Carl Zeiss
Stemi 2000-C stereomicroscope at magnication 2.25× equipped with a CMEX PRO-
5 DC.5000p digital camera and ImageFocus 4 software (M. Menchetti) and at 5×
magnication using Canon MP-E 65mm f/2.8 1–5× macro lens analysed with the
software ImageJ (Schneider et al. 2012) (E. Schifani).
We measured a total of 22 workers of L. maltaeus from Malta and Italy (Sicily
and Calabria) and 13 workers of L. emarginatus from Italy (Sicily, Aeolian Islands,
Calabria and Emilia-Romagna). We recorded six characters, including one chaeto-
taxonomic and four morphometric characters needed to distinguish L. emarginatus
from L. maltaeus according to Seifert (2020), and the cephalic size. e acronyms and
character denitions follow Seifert (2020):
CL: maximum cephalic length in median line; the head must be carefully tilted
to the position with the true maximum. Excavations of posterior head and/or
clypeus reduce CL.
CW: maximum cephalic width; this is either across, behind, or before the eyes.

Mattia Menchetti et al. / Journal of Hymenoptera Research 95: 129–142 (2023)
132
PoOc: postocular distance. Use a cross-scaled ocular micrometer and adjust the head
to the measuring position of CL. Caudal measuring point: median occipital
margin; frontal measuring point: median head at the level of the posterior eye
margin. Note that many heads are asymmetric and average the left and right
postocular distance.
MP6: length of the sixth (terminal) segment of maxillary palps.
nGen: with head in full face view, number of setae on head sides frontal of anterior
eye margin (gena). e bilateral sum is halved.
CS: arithmetic mean of CL and CW as less variable indicator of body size.
For all specimens we also calculated ratios (CL/CW, PoOC/CL and MP6/CL) and
the linear discriminant distinguishing L. maltaeus (D > 0) from L. cinereus Seifert, 1992,
L. emarginatus, L. illyricus and L. grandis (D < 0) at step 22a of the dichotomous key
by Seifert (2020): 45.42*PoOc/CL –0.183* CL/CW+55.63*MP6/CL +0.312*nGen
–25.59. Lasius emarginatus was distinguished from the other above-mentioned species
based on cuticle microsculpture, clypeal pubescence and setosity of the scapi following
Seifert (2020). All measurements are presented in mm.
Genetic analysis
A total of seven L. maltaeus and three L. emarginatus specimens belonging to dif-
ferent nests were selected for the genetic analysis. A few legs per specimens were
used. DNA-barcoding (mitochondrial gene cytochrome c oxidase I, COI, 658
bp) data was generated at two institutes: the Centre for Biodiversity Genomics,
University of Guelph, Canada, using the primers LepF1 and LepR1 (deWaard et
al. 2008); the Buttery Diversity and Evolution Lab (BDEL), following the proto-
col by Schär et al. (2020) and using the primers LCO1490/HC02198 (Folmer et
al. 1994). In the latter case, PCR products were visualized by gel electrophoresis
and sent to Macrogen Europe for Sanger sequencing. Raw sequences were edited
and aligned in Geneious Prime 2020.2.4 (Kearse et al. 2012). Chromatograms and
sequences have been inspected for the presence of, respectively, double peaks and
stop codons.
We also retrieved from GenBank a total of ten sequences of 658 bp identied
as L. emarginatus: one from Schär et al. (2018) (accession number LT977448), four
from Schär et al. (2020) (accession numbers MT606324, MT606325, MT606326,
and MT606327) and ve from Blatrix et al. (2020) (accession numbers MH138380,
MH138381, MH138384, MH138385, and MH138386). e haplotype network was
created using the program TCS 1.21 (Clement et al. 2000) and then graphically edited
with tcsBU (dos Santos et al. 2016) and Adobe Illustrator CC 2019. All the newly
generated sequences were submitted to GenBank (accession numbers OQ025622-
OQ025631, see also the Suppl. material 1: table S1) and to BOLD (dataset DS-ANT-
LMAL, doi: http://dx.doi.org/10.5883/DS-ANTLMAL).

Lasius maltaeus is not endemic to the Maltese Islands 133
Figure 1. Distribution of the samples of L. maltaeus (red) and L. emarginatus (yellow) investigated in this
study with quantitative morphology and/or molecular analysis. e shape of the points represents whether
the samples were identied with the linear discriminant (circles: ID in this study; triangles: ID by Seifert
2020; squares: no linear discriminant was used). Inner circles indicate whether molecular data (COI)
has been used (black circles: sequences generated in this study; white circles: sequences retrieved from
GenBank). Light blues lines in the map below indicate the coastline during the Last Glacial Maximum
(LGM). Main toponyms discussed in the text are highlighted in the map.

Mattia Menchetti et al. / Journal of Hymenoptera Research 95: 129–142 (2023)
134
Results
Samples identied by quantitative morphology and/or molecular analysis as L. maltaeus and
L. emarginatus were distributed allopatrically (Fig. 1). All samples from the Italian peninsula,
the Aeolian Islands and from north-eastern Sicily (Peloritani mountains and Etna) belonged
to L. emarginatus, while all samples from Malta and all other samples from Sicily (coming
from the north-western and south-eastern sectors) were identied as L. maltaeus (Fig. 1).
Morphological analysis
Quantitative morphological data are summarized in Table 1 and raw data is available in
the Suppl. material 1: table S2. All specimens were unambiguously classied as either
L. maltaeus or L. emarginatus according to the linear discriminant scores even without
considering nest means of multiple workers, while all individual characters overlapped
between the two species (Table 1, Figs 2, 3). For what concerns L. maltaeus, our data
slightly extend the minimum and maximum range of all investigated characters except
for the maximum range of PoOC and CW.
Table 1. Summary of the morphological dierences between L. maltaeus (n=22) and L. emarginatus
(n=13) specimens measured in this study. All morphometric characters are reported in mm as mean
± standard deviation (minimum, maximum). e raw data is available in Suppl. material 1: table S2.
L. maltaeus L. emarginatus
CL 0.96 ± 0.07 (0.79, 1.11) 0.99 ± 0.05 (0.91, 1.05)
CW 0.9 ± 0.07 (0.73, 1.03) 0.93 ± 0.07 (0.82, 1.02)
PoOC 0.22 ± 0.02 (0.17, 0.25) 0.22 ± 0.02 (0.19, 0.25)
MP6 0.22 ± 0.02 (0.18, 0.26) 0.21 ± 0.01 (0.18, 0.23)
nGen 13.36 ± 2.45 (8, 18) 7.04 ± 1.83 (4, 10)
CS 0.93 ± 0.07 (0.76, 1.07) 0.96 ± 0.05 (0.87, 1.03)
CL/CW 1.07 ± 0.03 (1.01, 1.14) 1.07 ± 0.07 (1.01, 1.25)
PoOC/CL 0.23 ± 0.01 (0.20, 0.25) 0.22 ± 0.01 (0.21, 0.24)
MP6/CL 0.23 ± 0.02 (0.21, 0.26) 0.21 ± 0.01 (0.2, 0.22)
D 1.91 ± 0.83 (0.76, 3.92) -1.35 ± 0.84 (-3.17, -0.17)
Figure 2. Frontal view of the head and lateral view of a L. maltaeus worker from Sicily (voucher
ES16A036).

Lasius maltaeus is not endemic to the Maltese Islands 135
Genetic analysis
We generated ten COI sequences with a length of 658 bp. e haplotype network
analysis (Fig. 4) based on a total of 20 COI sequences (8 for L. maltaeus and 12 for
L. emarginatus) show two distinct haplogroups that match the morphological spe-
cies identication. Within the L. maltaeus haplogroup, the two L. maltaeus colonies
analysed from Malta had distinct haplotypes, both shared with the Sicilian popula-
tions: one shares the haplotype with a colony from the Hyblean Plateau (voucher
MM21B061a1 from Cavagrande del Cassibile, Siracusa) and the other with samples
from the mountains in the area of Palermo (voucher MM18A053a1 from Monte
Moarda and sequence MT606327 from Monte Pellegrino). A total of ve haplotypes
were found in L. maltaeus, with a maximum intraspecic mtDNA divergence of 0.6%,
while in L. emarginatus we found six haplotypes and a maximum intraspecic mtDNA
divergence of 1.1%. e minimum and maximum interspecic mtDNA divergence
found were, respectively, 2.9% and 3.8%.
Figure 3. Morphological dierences between L. maltaeus (red) and L. emarginatus (yellow) specimens
according to cephalic size (CS) and the linear discriminant D from Seifert (2020) (green dashed line: < 0
L. emarginatus, > 0 L. maltaeus). Circles represent the specimens measured in this study, while triangles
indicate the specimens from Seifert (2020). Sequenced specimens present an inner circle, which is black if
generated in this study or white if obtained from GenBank (i.e. from Schär et al. 2020). Note that indi-
vidual specimens may be misidentied by the discriminant scores, but not nest means of multiple workers
according to the data provided by Seifert (2020).

Mattia Menchetti et al. / Journal of Hymenoptera Research 95: 129–142 (2023)
136
Discussion
Quantitative morphology and mtDNA data agree and allow for the unambiguous
distinction of L. emarginatus from L. maltaeus. Our results demonstrate that the range
of the latter mostly resides in Sicily (where it shows the larger haplotype diversity),
making it a Siculo-Maltese endemic rather than a Maltese endemic species. e long
history of doubts over the identity of the Maltese populations, culminating with the
description of L. maltaeus (Schembri and Collingwood 1981,1995; Seifert 2020), per-
haps reects the great interest of naturalists for the biodiversity of smaller islands, while
the Sicilian populations were long overlooked. As a consequence of this discovery,
at least two species of the L. emarginatus complex inhabit Italy, while further inves-
tigation should check for the possible presence of L. illyricus (Schifani 2022). As a
result, L. balearicus Talavera, Espadaler & Vila, 2015 from Mallorca (Balearic Islands),
L. cypereus Seifert, 2020 from Cyprus, and L. kritikos Seifert, 2020 and L. tapinomoides
Salata & Borowiec, 2018 from Crete are the only Lasius species known to occur in a
single island (Talavera et al. 2015; Salata and Borowiec 2018; Seifert 2020).
e vicariance between L. emarginatus and L. maltaeus in the broader context of the
Siculo-Maltese archipelago and Italian peninsula follows a fascinating biogeographic
pattern that reects the complex paleogeographic history of the region. e region of
Trapani and Palermo Mountains in north-western Sicily, and the Hyblaean plateau in
south-eastern Sicily (recurrently linked to Malta by land bridges), represent the two
most ancient sectors of the island to have emerged from the sea perhaps even before
the upper Pliocene (Masini and Sarà 1998; Guarino and Pasta 2018). Both regions are
recognized as well-dened biogeographic provinces hosting a signicant number of
endemic fauna and ora (Brullo et al. 1995, 2011; Guarino and Pasta 2018; Schifani
Figure 4. Haplotype network of L. maltaeus and L. emarginatus mitochondrial COI sequences. Colour
and size of the circles indicate geographical origin and number of the samples, respectively.

Lasius maltaeus is not endemic to the Maltese Islands 137
et al. 2020; Schmitt et al. 2021). On the other hand, the Etna and Peloritani regions
of north-eastern Sicily, have a distinct geology and paleogeographic history and appear
biogeographically more similar to the Italian peninsula or other Mediterranean re-
gions: the Etna is fairly recent, emerging only about 570 thousand years ago, while the
Peloritani alongside Calabria rotated counterclockwise from the Sardinian-Corsican
microplate into their current position during the Alpine Orogeny (Stöck et al. 2008;
Sciandrello et al. 2015; Scalercio et al. 2020; Schmitt et al. 2021).
e fauna and ora of north-western and south-eastern Sicily are more inuenced
by colonization from the Africa’s Maghreb region compared to the north-east, which
hosts more species from continental Europe (Masini and Sarà 1998; Stöck et al. 2008;
Sciandrello et al. 2015; Alicata and Schifani 2019; Schifani et al. 2020, 2022a, b).
e case of the green toads Bufotes boulengeri siculus (Stöck et al., 2008) and Bufotes
viridis balearicus (Boettger, 1880) is particularly striking as the distribution of these
two subspecies (the rst of Maghrebian origin, the second one European) mirrors
those of L. maltaeus and L. emarginatus respectively (Stöck et al. 2008; Dufresnes et
al. 2019). Among other ants, the distribution of Aphaenogaster trinacriae Alicata &
Schifani, 2019 resembles that of L. maltaeus within Sicily, while Formica clara Forel,
1886 and Solenopsis orbula Emery, 1875 may be restricted to the north-east of the
island (Alicata and Schifani 2019; Schifani et al. 2021, 2022b). However, western
and southern Sicily may also have acted as a refugium for relict European lineages, as
recently shown among butteries (Scalercio et al. 2020). Based on the available mor-
phological and molecular data, it is possible that the sister species of L. maltaeus is the
European L. emarginatus, but phylogenetic evidence for a broader number of species
(e.g., including the Maghrebian L. tebessae) is needed to conrm this.
e ant faunas of Malta and Sicily share most species and the Siculo-Maltese archi-
pelago may be considered as a single bioregion (Wang et al. 2022). e observation that
the Maltese and Sicilian populations of L. maltaeus are not genetically dierentiated and
share two dierent COI haplotypes, suggest recent exchanges between the two (involving
multiple queens), which is congruent with the hypothesis that the species exploited land
bridges during the last glacial period. Beyond the case of L. maltaeus, our results question
the existence of Maltese endemic ants. While Lasius maltaeus becomes the fourth Siculo-
Maltese endemic species after Aphaenogaster orii Emery, 1915, Temnothorax lagrecai
(Baroni Urbani, 1964), and Temnothorax marae Alicata, Prebus & Schifani, 2022 (Ali-
cata and Schifani 2019; Schifani et al. 2022c), there are three remaining species which are
currently considered Maltese endemics: Aphaenogaster melitensis Santschi, 1933, Strongy-
lognathus insularis Baroni Urbani, 1968, and Temnothorax splendidiceps (Baroni Urbani,
1968) (Baroni Urbani 1968; Boer 2013). Each of them belongs to a taxonomically unre-
solved species group and their validity should be reassessed. In particular, the characters
allegedly distinguishing S. insularis from its southern Italian counterpart S. destefanii
Emery, 1915 were never quantied (Baroni Urbani 1968; Sanetra et al. 1999). A similar
situation occurs with T. splendidiceps and the Sicilian endemic T. laestrygon (Santschi,
1931), with the dierence that the species status of both is poorly supported because
no character has been described to distinguish them from T. exilis in its current concept

Mattia Menchetti et al. / Journal of Hymenoptera Research 95: 129–142 (2023)
138
(Baroni Urbani 1968; Salata et al. 2018). Finally, concerning A. melitensis, some mor-
phological data are available, but the identity of the Sicilian morphotypes corresponding
to the old A. ionia concept requires further investigation (authors’ unpublished data) and
the whole group awaits a comprehensive revision (Schifani et al. 2022d).
Estimated speciation times for ants (and especially Formicinae, see Schär et al.
2018, 2020) are on average longer than in many other insect groups and the short
time of separation between Malta and Sicily suggests allopatric speciation of isolated
Maltese populations to be unlikely.
Acknowledgements
We wish thank Antonio Adorno, Simone Costa, Emanuele Genduso, Roberto Ri-
trovato, Norian Saliba and Roberto Viviano for the specimens provided, and Cecíl-
ia Corbella for help in the laboratory. We thank Sebastian Salata, iago Silva, one
anonymous reviewer and the editor Francisco Hita-Garcia for their comments on an
earlier version of this manuscript. Support for this research was provided by ‘La Caixa’
Foundation (ID 100010434) to Mattia Menchetti (grant LCF/BQ/DR20/11790020).
Morphological data, specimen collection data and GenBank accession numbers are
available in the Suppl. material 1. Sequences are also available in the BOLD dataset
DS-ANTLMAL (doi: http://dx.doi.org/10.5883/DS-ANTLMAL).
References
Alicata A, Schifani E (2019) ree endemic Aphaenogaster from the Siculo-Maltese archipelago
and the Italian Peninsula: part of a hitherto unrecognized species group from the Maghreb?
(Hymenoptera: Formicidae: Myrmicinae). Acta Entomologica Musei Nationalis Pragae
59(1): 1–16. https://doi.org/10.2478/aemnp-2019-0001
Baroni Urbani C (1968) Studi sulla mirmecofauna d’Italia. IV. La fauna mirmecologica delle
isole Maltesi ed il suo signicato ecologico e biogeograco. Annali del Museo Civico di
Storia Naturale “Giacomo Doria” 77: 408–559. https://doi.org/10.5281/zenodo.26774
Blatrix R, Aubert C, Decaens T, Berquier C, Andrei-Ruiz MC, Galkowski C (2020) Contribution
of a DNA barcode to an assessment of the specicity of ant taxa (Hymenoptera: Formicidae)
on Corsica. European Journal of Entomology 117: 420–429. https://doi.org/10.14411/
eje.2020.046
Bolton B 2022 An online catalog of the ants of the world. https://antcat.org [accessed
15.10.2022]
Borowiec L, Salata S (2013) Ants of Greece – additions and corrections (Hymenoptera:
Formicidae). Genus 24(3–4): 335–401. https://doi.org/10.5281/zenodo.11430
Boudinot BE, Borowiec ML, Prebus MM (2022) Phylogeny, evolution, and classication of the
ant genus Lasius, the tribe Lasiini and the subfamily Formicinae (Hymenoptera: Formicidae).
Systematic Entomology 47(1): 113–151. https://doi.org/10.1111/syen.12522

Lasius maltaeus is not endemic to the Maltese Islands 139
Brullo S, Minissale P, Spampinato G (1995) Considerazioni togeograche sulla ora della
Sicilia. Ecologia Mediterranea 21: 99–117. https://doi.org/10.3406/ecmed.1995.1759
deWaard JR, Ivanova NV, Hajibabaei M, Hebert PDN (2008) Assembling DNA barcodes. In:
Martin CC, Martin CC (Eds) Environmental Genomics. Methods in Molecular Biology,
vol 410. Humana Press, Totowa & New Jersey, 275–294. https://doi.org/10.1007/978-1-
59745-548-0_15
Dufresnes C, Mazepa G, Jablonski D, Oliveira RC, Wenseleers T, Shabanov DA, Auer M, Ernst
R, Koch C, Ramírez-Chaves HE, Mulder KP, Simonov E, Tiutenko A, Kryvokhyza D,
Wennekes PL, Oleksandr IZ, Korshunov OV, Al-Johany AM, Peregonstev EA, Masroor
R, Betto-Colliard C, Denoël M, Borkin LJ, Skorinov DV, Pasynkova RA, Mazanaeva LF,
Rosanov JM, Dubey S, Litvinchuk S (2019) Fifteen shades of green: the evolution of
Bufotes toads revisited. Molecular Phylogenetics and Evolution 141: 106615. https://doi.
org/10.1016/j.ympev.2019.106615
Fattorini S (2011) Biogeography of tenebrionid beetles (Coleoptera: Tenebrionidae) in
the circum-Sicilian islands (Italy, Sicily): Multiple biogeographical patterns require
multiple explanations. European Journal of Entomology 108(4): 659–672. https://doi.
org/10.14411/eje.2011.084
Foglini F, Prampolini M, Micallef A, Angeletti L, Vandelli V, Deidun A, Soldati M, Taviani
M (2016) Late Quaternary coastal landscape morphology and evolution of the Maltese
Islands (Mediterranean Sea) reconstructed from high-resolution seaoor data. Geological
Society, London, Special Publications 411: 77–95. https://doi.org/10.1144/SP411.12
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplication
of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.
Molecular Marine Biology and Biotechnology 3: 294–299. https://pubmed.ncbi.nlm.nih.
gov/7881515
Guarino R, Pasta S (2018) Sicily: the island that didn’t know to be an archipelago. Berichte Der
Reinhold-Tuxen-Gesellschaft 30: 133–148. https://iris.unipa.it/handle/10447/335722
Hölldobler B, Wilson EO (1990) e ants. Harvard University Press, Oxford, USA, 746 pp.
https://doi.org/10.1007/978-3-662-10306-7
Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A,
Markowitz S, Duran C, ierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious
Basic: an integrated and extendable desktop software platform for the organization and
analysis of sequence data. Bioinformatics 28(12): 1647–1649. https://doi.org/10.1093/
bioinformatics/bts199
Maruyama M, Steiner FM, Stauer C, Akino T, Crozier RH, Schlick-Steiner BC (2008) A DNA
and morphology based phylogenetic framework of the ant genus Lasius with hypotheses for
the evolution of social parasitism and fungiculture. BMC evolutionary biology 8(1): 1–15.
https://doi.org/10.1186/1471-2148-8-237
Masini F, Sarà M (1998) Asoriculus burgioi sp. nov. (Soricidae, Mammalia) from the Monte
Pellegrino faunal complex (Sicily). Acta Zoologica Cracoviensis 41: 111–124. http://www.
isez.pan.krakow.pl/journals/azc/pdf/azc_v/41(1)/41(1)_10.pdf
Médail F (2022) Plant biogeography and vegetation patterns of the Mediterranean islands. e
Botanical Review 88: 1–67. https://doi.org/10.1007/s12229-021-09245-3

Mattia Menchetti et al. / Journal of Hymenoptera Research 95: 129–142 (2023)
140
Salata S, Borowiec L (2018) A new species of the ant genus Lasius Fabricius, 1804 from
Crete (Hymenoptera, Formicidae). ZooKeys 789: 139–159. https://doi.org/10.3897/
zookeys.789.27022
Salata S, Borowiec L, Trichas A (2018) Taxonomic revision of the Cretan fauna of the genus
Temnothorax Mayr, 1861 (Hymenoptera: Formicidae), with notes on the endemism of ant
fauna of Crete. Annales Zoologici (Warsaw) 68(4): 769–808. https://doi.org/10.3161/00
034541ANZ2018.68.4.004
Sanetra M, Güsten R, Schulz A (1999) On the taxonomy and distribution of Italian
Tetramorium species and their social parasites (Hymenoptera Formicidae). Memorie della
Società Entomologica Italiana 77: 317–357. https://www.antcat.org/references/132198
Salvi D, Schembri PJ, Sciberras A, Harris DJ (2014) Evolutionary history of the Maltese wall
lizard Podarcis lfolensis: insights on the ‘Expansion–Contraction’ model of Pleistocene
biogeography. Molecular Ecology 23(5): 1167–1187. https://doi.org/10.1111/mec.12668
Scalercio S, Cini A, Menchetti M, Vodă R, Bonelli S, Bordoni A, Casacci LP, Dincă V, Balletto
E, Vila R, Dapporto L (2020) How long is 3 km for a buttery? Ecological constraints and
functional traits explain high mitochondrial genetic diversity between Sicily and the Italian
Peninsula. Journal of Animal Ecology 89(9): 2013–2026. https://doi.org/10.1111/1365-
2656.13196
Schär S, Talavera G, Espadaler X, Rana JD, Andersen Andersen A, Cover SP, Vila R (2018)
Do Holarctic ant species exist? Trans‐Beringian dispersal and homoplasy in the Formicidae.
Journal of Biogeography 45(8): 1917–1928. https://doi.org/10.1111/jbi.13380
Schär S, Menchetti M, Schifani E, Hinojosa JC, Platania L, Dapporto L, Vila R (2020)
Integrative biodiversity inventory of ants from a Sicilian archipelago reveals high diversity
on young volcanic islands (Hymenoptera: Formicidae). Organisms Diversity & Evolution
20(3): 405–416. https://doi.org/10.1007/s13127-020-00442-3
Schär S, Talavera G, Rana JD, Espadaler X, Cover SP, Shattuck SO, Vila R (2022) Integra-
tive taxonomy reveals cryptic diversity in North American Lasius ants, and an overlooked
introduced species. Scientic Reports 12(1): 1–12. https://doi.org/10.1038/s41598-022-
10047-9
Schembri SP, Collingwood CA (1981) A revision of the Myrmecofauna of the Maltese Islands
(Hymenoptera, Formicidae). Annali del Museo Civico di Storia Naturale “Giacomo Doria”
83: 417–442. https://doi.org/10.5281/zenodo.24597
Schembri SP, Collingwood CA (1995) e myrmecofauna of the Maltese Islands. Remarks and
additions (Hymenoptera Formicidae). Bollettino della Società Entomologica Italiana 127:
153–158. https://www.antwiki.org/wiki/images/6/66/Schembri_Collingwood_1995.pdf
Schifani E, Massa B (2020) First record of Lasius illyricus Zimmermann, 1935 (Hymenop-
tera, Formicidae) from Armenia. Far Eastern Entomologist 398: 24–28. https://doi.
org/10.25221/fee.398.4
Schifani E, Scupola A, Alicata A (2020) Morphology, ecology and biogeography of Myrmecina
sicula André, 1882, rediscovered after 140 years (Hymenoptera, Formicidae). Biogeographia
35: 105–116. https://doi.org/10.21426/B635048444
Schifani E (2022) e new Checklist of the Italian Fauna: Formicidae. Biogeographia 37:
ucl006. https://doi.org/10.21426/B637155803

Lasius maltaeus is not endemic to the Maltese Islands 141
Schifani E, Giannetti D, Csősz S, Castellucci F, Luchetti A, Castracani C, Spotti Fa, Mori
A, Grasso DA (2022a) Is mimicry a diversication-driver in ants? Biogeography,
ecology, ethology, genetics and morphology dene a second West-Palaearctic Colobopsis
species (Hymenoptera: Formicidae). Zoological Journal of the Linnean Society 194(4):
1424–1450. https://doi.org/10.1093/zoolinnean/zlab035
Schifani E, Alicata A, Menchetti M (2022b) Following the Apennines: updating the distribution
of Formica clara and F. rubarbis in Italy (Hymenoptera, Formicidae). Biogeographia 37:
a014. https://doi.org/10.21426/B637155107
Schifani E, Prebus MM, Alicata A (2022c) Integrating morphology with phylogenomics to
describe four island endemic Temnothorax species from Sicily and Malta (Hymenoptera,
Formicidae). European Journal of Taxonomy 833: 143–179. https://doi.org/10.5852/
ejt.2022.833.1891
Schifani E, Alicata A, Menchetti M, Borowiec L, Fisher BL, Karaman C, Kiran K, Oueslati W,
Salata S, Blatrix R (2022d) Revisiting the morphological species groups of West-Palearctic
Aphaenogaster ants (Hymenoptera: Formicidae) from a phylogenetic perspective: toward an
evolutionary classication. Arthropod Systematics & Phylogeny 80: 627–648. https://doi.
org/10.3897/asp.80.e84428
Schlick-Steiner BC, Steiner FM, Schödl S, Seifert B (2003) Lasius austriacus sp. n., a Central
European ant related to the invasive species Lasius neglectus. Sociobiology 41(3): 725–736.
http://antbase.org/ants/publications/20243/20243.pdf
Sciandrello S, Guarino R, Minissale P, Spampinato G (2015) e endemic vascular ora
of Peloritani Mountains (NE Sicily): Plant functional traits and phytogeographical
relationships in the most isolated and fragmentary micro-plate of the Alpine orogeny. Plant
Biosystems 149(5): 838–854. https://doi.org/10.1080/11263504.2014.908978
Schmitt T, Fritz U, Delno M, Ulrich W, Habel JC (2021) Biogeography of Italy revisited:
genetic lineages conrm major phylogeographic patterns and a pre-Pleistocene origin of
its biota. Frontiers in Zoology 18(1): 1–13. https://doi.org/10.1186/s12983-021-00418-9
Seifert B (1991) Lasius platythorax n. sp., a widespread sibling species of Lasius niger
(Hymenoptera: Formicidae). Entomologia Generalis 16(1): 69–81. https://doi.
org/10.1127/entom.gen/16/1991/69
Seifert B (1992) A taxonomic revision of the Palaearctic members of the ant subgenus Lasius
s. str. (Hymenoptera, Formicidae). Abhandlungen und Berichte des naturkundemuseums
Görlitz 66: 1–67. https://doi.org/10.5281/zenodo.24614
Seifert B (2018) e Ants of Central and North Europe. Lutra Verlags- und Vertriebsgesellschaft,
Tauer, Germany, 408 pp.
Seifert B (2020) A taxonomic revision of the Palaearctic members of the subgenus Lasius s.
str. (Hymenoptera, Formicidae). Soil Organisms 92(1): 15–86. https://doi.org/10.25674/
so92iss1pp15
Seifert B, Galkowski C (2016) e Westpalaearctic Lasius paralienus complex (Hymenoptera:
Formicidae) contains three species. Zootaxa 4132(1): 44–58. https://doi.org/10.11646/
zootaxa.4132.1.4
Stöck M, Sicilia A, Belore NM, Buckley D, Lo Brutto S, Lo Valvo M, Arculeo M (2008)
Post-Messinian evolutionary relationships across the Sicilian channel: mitochondrial and

Mattia Menchetti et al. / Journal of Hymenoptera Research 95: 129–142 (2023)
142
nuclear markers link a new green toad from Sicily to African relatives. BMC Evolutionary
Biology 8(1): 1–19. https://doi.org/10.1186/1471-2148-8-56
Talavera G, Espadaler X, Vila R (2015) Discovered just before extinction? e rst endemic
ant from the Balearic Islands (Lasius balearicus sp. nov.) is endangered by climate change.
Journal of Biogeography 42(3): 589–601. https://doi.org/10.1111/jbi.12438
ake MA (1985) e biogeography of the Maltese islands, illustrated by the Clausiliidae.
Journal of Biogeography 12(3): 269–287. https://doi.org/10.2307/2844999
Van Loon AJ, Boomsma JJ, Andrasfalvy A (1990) A new polygynous Lasius species
(Hymenoptera; Formicidae) from central Europe. Insectes Sociaux 37(4): 348–362.
https://doi.org/10.1007/BF02225997
Wang R, Kass JM, Galkowski C, Garcia F, Hamer MT, Radchenko A, Salata S, Schifani E,
Yusupov ZM, Economo EP, Guénard B (2022) Geographic and climatic constraints on
bioregionalization of European ants. Journal of Biogeography. https://doi.org/10.1111/
jbi.14546
Wilson EO (1955) A monographic revision of the ant genus Lasius. Bulletin of the museum
of comparative Zoology at Harvard College 113: 1–201. https://doi.org/10.5281/zeno-
do.25290
Supplementary material 1
e collecting data and voucher identiers of the specimens
Authors: Mattia Menchetti, Enrico Schifani, Antonio Alicata, Roger Vila
Data type: morphological data, specimen collection data, GenBank accession numbers
Explanation note: e Suppl. material presents the collecting data and voucher
identiers of the specimens we analysed as well as the morphological data and the
GenBank accession numbers of the sequences produced/analysed in this study.
Copyright notice: is dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). e Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/jhr.95.96365.suppl1