Insights into the evolution, biogeography and natural history of the acorn ants, genus Temnothorax Mayr (hymenoptera: Formicidae)

Background Temnothorax (Formicidae: Myrmicinae) is a diverse genus of ants found in a broad spectrum of ecosystems across the northern hemisphere. These diminutive ants have long served as models for social insect behavior, leading to discoveries about social learning and inspiring hypotheses about the process of speciation and the evolution of social parasitism. This genus is highly morphologically and behaviorally diverse, and this has caused a great deal of taxonomic confusion in recent years. Past efforts to estimate the phylogeny of this genus have been limited in taxonomic scope, leaving the broader evolutionary patterns in Temnothorax unclear. To establish the monophyly of Temnothorax, resolve the evolutionary relationships, reconstruct the historical biogeography and investigate trends in the evolution of key traits, I generated, assembled, and analyzed two molecular datasets: a traditional multi-locus Sanger sequencing dataset, and an ultra-conserved element (UCE) dataset. Using maximum likelihood, Bayesian, and summary-coalescent based approaches, I analyzed 22 data subsets consisting of 103 ingroup taxa and a maximum of 1.8 million base pairs in 2485 loci. Results The results of this study suggest an origin of Temnothorax at the Eocene-Oligocene transition, concerted transitions to arboreal nesting habits in several clades during the Oligocene, coinciding with ancient global cooling, and several convergent origins of social parasitism in the Miocene and Pliocene. As with other Holarctic taxa, Temnothorax has a history of migration across Beringia during the Miocene. Conclusions Temnothorax is corroborated as a natural group, and the notion that many of the historical subgeneric and species group concepts are artificial is reinforced. The strict form of Emery’s Rule, in which a socially parasitic species is sister to its host species, is not well supported in Temnothorax. Electronic supplementary material The online version of this article (10.1186/s12862-017-1095-8) contains supplementary material, which is available to authorized users.

petiole combined with a broad, campaniform post-petiole in dorsal view and a dorsally contiguous, often rounded, mesosoma in profile. Some additional characters that are associated with this syndrome, but not consistently present, are enlarged, spindle-shaped middle and hind femora, and iridescently colored integument. Mann [16] split Macromischa into three subgenera to accommodate some of the variation in the group, erecting the subgenera Croesomyrmex and Antillaemyrmex. Later, Brown [17] provisionally synonymized Macromischa with Leptothorax, but this name was still in use as a subgenus when Baroni Urbani [18] postulated that Macromischa was probably polyphyletic because many of the features that define this syndrome are found independently among clearly different lineages of Temnothorax. This taxon name fell out of use when it was formerly synonymized with Leptothorax by Snelling [19], who observed that there are many Central American species that are morphologically intermediate between Leptothorax and Macromischa.
Several subgenera have been proposed specifically for Temnothorax: Emery [20] erected the subgenus Dichothorax to accommodate the morphologically aberrant ground-nesting Nearctic species T. pergandei [ Figure 1c]. This species differs from all others in the Nearctic region by its strongly depressed propodeum, massive, rounded promesonotum, and elongate petiole. Several authors have noted that this morphology is also present in several species around the Palearctic Mediterranean and North Africa, namely T. recedens [ Figure 1d] and T. schaufussi [21,22]. Ashmead [22] raised Dichothorax to genus level, but this change was not widely adopted; Hamman & Klemm [23] proposed Icothorax for the African species T. megalops [ Figure 1e]; both names were used as subgenera until Bolton [24] synonymized them with Leptothorax. Similarly, Menozzi [25] created the subgenus Myrmammophilus to accommodate the Mediterranean T. finzii [ Figure 1f] because the reproductives lacked a discoidal cell in their forewing, a feature that, in the light of the present study, appears to be plastic within Temnothorax and the LGG. This species is not particularly morphologically divergent when the diversity of North African species is considered, and appears to bear close affinities to the laurae species group, although it was placed in the recedens-group by Salata & Borowiec [26].
As the passages above indicate, the taxonomic history of Temnothorax is convoluted: senior synonym of no less than thirteen proposed genera and subgenera, the current definition was only arrived at in 2015 [13], when the species of the erstwhile socially parasitic genera Chalepoxenus [ Figure 4d], Myrmoxenus [ Figure 4b] and Protomognathus [ Figure 4a] were synonymized with Temnothorax based on molecular evidence (see [27,28] for an extensive discussion). Bolton [1,24] did the bulk of the work updating Temnothorax to its modern definition, however, by formally designating the names Antillaemyrmex, Croesomyrmex, Dichothorax, Icothorax, Macromischa, Myrafant, and Myrmammophilus as junior synonyms of Leptothorax and later Temnothorax. Moreover, Bolton [24] provided morphological diagnoses to differentiate Temnothorax, the LGG, and the morphologically convergent Nesomyrmex [1, Figure 2b] based on mouthpart morphology.
The disproportionate number of species among these biogeographic areas may be the product of several factors, namely the lack of a large-scale systematic synopsis in the Palearctic, where the large number of small-scale regional revisions has likely inflated the number of species due to undetected synonymies, and the relative paucity of collection efforts in Northern and Central Mexico where Temnothorax diversity is likely to be very high based on the number of undescribed species from southern Arizona (Stefan Cover, pers. comm.). Additionally, recent collecting initiatives in Central America, the Baja California peninsula, and the Dominican Republic have recovered many new species, which are currently in the process of being formally described (Prebus in prep.). Many of the authors cited above have offered informal species-group descriptions, but a formal systematic synthesis has not yet been attempted for any biogeographical region, much less on a global scale.

Fossil record
The fossil record for Temnothorax includes one species from Dominican Amber (15)(16)(17)(18)(19)(20) [41,42, but see 43], six from Baltic Amber (38)(39)(40)(41)(42)(43)(44)(45)(46)(47) [44][45][46][47][48], and eight undescribed species from various European late Eocene and late Oligocene amber deposits (23-47 Ma) [49][50][51]. Temnothorax praecreolus de Andrade 1992, from the Dominican amber, is known from a single worker that exhibits several of the features associated with the Macromischa syndrome, including an elongate petiole, a broad, campaniform post-petiole, and enlarged mid and hind femora. While somewhat dorso-ventrally compressed, this fossil still possesses a remarkable number of visible characters. I obtained this fossil for inspection from the Naturhistoriches Museum, Basel, Switzerland [ Figure 5a]. Mayr [44] described four species of Macromischa and one Leptothorax species, L. gracilis from Baltic amber [ Figure 5d]. Wheeler [53] recognized one of Mayr's Macromischa species as Vollenhovia, and erected a new genus for the remainder of them, Nothomyrmica. Wheeler did not explicitly state his reasoning for this transfer, implying that because these species possess only one of the features of the Macromischa syndrome, a pedunculate petiole, that this precludes them from placement in any known genus. Wheeler's [53] treatment also introduced four new fossil species of Leptothorax from Baltic amber, noting that several of these species bear a close resemblance to L. acervorum in general bauplan, but four of the five fossil species have 12-merous antennae, a condition not found in the extant Leptothorax s.s.. Bolton [1] transferred all five of the fossil Lepthothorax species to Temnothorax.
More recently, Dlussky & Radchenko [54] dismantled Nothomyrmica, transferring N. petiolata to Temnothorax [ Figure 5b], designating a new genus, Eocenomyrma [ Figure 5c], for N. rugostriata and three newly described fossil species, and subsequently synonymized the remainder of Nothomyrmica with Myrmica [55]. Dlussky & Radchenko [54] state that Eocenomyrma, while clearly separable from Temnothorax by clypeal structure, is probably a member the tribe Formicoxenini (sensu Bolton [1]). This same treatment did not give an explicit reason for transferring N. petiolata, only stating that it is 'indistinguishable by all its characteristic features' from Temnothorax, without enumerating the characters that lead them to this conclusion. Temnothorax petiolata bears long propodeal spines, 12-merous antennae, a metanotal groove, and a pedunculate petiole. These features in combination do indeed suggest that this species cannot be excluded from the crown group of Temnothorax, but I am hesitant to make a positive placement due to the lack of information about mandibular dentition and mouthpart morphology. For example, T. petiolata also closely resembles extant species of the Indomalayan and Australasian genus Vombisidris Bolton [56], which recent molecular work has shown to be a close relative of [Temnothorax + LGG] [13]. Vombisidris has unique mandibular morphology, with a large diastema between the preapical mandibular tooth and the basalmost three. I have not yet been able to personally inspect the Baltic Amber fossils of Temnothorax, although images of T. gracilis and T. petiolata are available on AntWeb (http://www.antweb.org) [ Figure 5b and d]. In a personal communication with Phillip Barden at the American Museum of Natural History, I inspected images of a Baltic Amber worker specimen identified as Temnothorax which had 12 antennomeres and a crest on the maxillary stipes, a condition not known from any extant Temnothorax or LGG species [ Figure 4d]. This casts considerable doubt on whether Temnothorax fossils from Baltic Amber have been accurately identified. Consequently, I conservatively treat all Baltic Amber fossils currently classified as Temnothorax as stem lineages of the clade [Temnothorax + LGG] until the mouthpart morphology of each described fossil species can be inspected.

Taxonomic results
The constrained analysis of the Sanger sequencing dataset shown in Figure [ 2] in the main article. Temnothorax is composed of seven well-supported major clades in a pectinate series, which display a high degree of biogeographic structure. Each clade generally corresponds to a clade in the unconstrained Sanger dataset analyses Additional File [3], which is noted at the beginning of the description. Support values are given in posterior probability (PP) from the MrBayes analysis, and in bootstraps (BS) from the IQTREE analysis.

andrei-clade
The exclusively western and southwestern North American andrei-clade forms the first group in the pectinate series, sister to all other crown Temnothorax. This clade is composed of the andrei-, nitens-, as well as T. gallae and T. nevadensis of the tricarinatus-group proposed by Mackay [29]. The tricarinatus-group is found to be paraphyletic: T. nevadensis and T. gallae are more closely related to T. andrei than they are to T. tricarinatus, which is nested within the salleiclade (see below). T. nitens is found to be sister to the andrei-group, but with low support (PP 1; BS 68).

sallei-clade
The sallei-clade, found in the southern Nearctic, Neotropics and the Greater Antilles, is the next to branch off. This group shows within-clade geographic structure, with the sallei and iris subclades distributed among the Greater Antilles and, in the case of T. allardycei, southern Florida and the Bahamas. These Caribbean species were informally classified by Baroni Urbani [18] as being members of the Macromischa allardycei-(T. allardycei), sallei-(T. laetus and T. sallei), splendens-(T. splendens), iris-(T. gundlachi), and versicolor-(T. poeyi) species groups. The sallei-group appears to be paraphyletic, with members distributed between the purpuratusand salvini-clades (see below). T. mmp11 and T. cf. striatulus form a third distinct group, known only from the cloud forests of Meso-America. The latter is, at first glance, indistinguishable from T. striatulus, but the comparative length of the petiolar peduncle and appendages neatly separate the two taxa. The fourth subgroup within the purpuratus-clade is the remainder of the tricarinatus-group proposed in Mackay [29] (T. carinatus, T. neomexicanus, T. obliquicanthus, T. punctithorax, T. cf. rugithorax, T. stenotyle, and T. tricarinatus), within which the striatulusand at least one member of the andersoni-species groups arose. This group is broadly distributed throughout the southern Nearctic, with at least two species (T. striatulus and T. mmp08) in the cloud forests of Meso-America. Following the purpuratus-clade, two smaller clades emerge.

rottenbergi-clade
The first of the smaller clades is the Palearctic rottenbergi-clade, composed of members of the nominal species group sensu Cagniant & Espadaler [30] (T. cabrerae, T. formosus, and T. semiruber), which are large, typically ground-nesting species distributed throughout the Mediterranean region, including North Africa and the Canary Islands. Two other species with a Mediterranean distribution are found to be affiliated with the rottenbergi-group: T. cf. flavispinus of the arboreal flavispinus-group (sensu Cagniant & Espadaler [30]) and the ground-nesting T. cristinae, which has been proposed to be closely related to T. exilis [57].

rugatulus-clade
The second of the smaller clades is the rugatulus-clade, which contains a mixture of Palearctic and broadly distributed North American species. The arboreal species T. clypeatus and T. corticalis (clypeatus-and corticalis-groups sensu Radchenko [32]), which are both rarely collected but apparently widely distributed in the western Palearctic, are contained within this group. The nominal species of this clade is commonly encountered nesting under stones in montane western North America (longispinosus group sensu Mackay [29]), sister to an undescribed species collected from a mesic mountaintop habitat on the southern Baja California peninsula. T. schaumii (schaumii-group Mackay [29]) and T. smithi (silvestrii-group Mackay [29]) are somewhat rarely collected arboreal species from eastern North America. The longispinosus-and schaumii-groups are polyphyletic as originally described: T. rugatulus and T. schaumii are more closely related to each other than to the other members of the original groups, all of which are monophyletic and nested within the Palearctic-clade (see below).

salvini-clade
The Neotropical and Caribbean salvini-clade is the next group to emerge, followed by the obturator and Palearctic-clades. The members of the salvini-clade are morphologically and behaviorally diverse, including many arboreal species from Meso-America and the enigmatic ground-nesting eastern North American species T. pergandei (formerly subgenus Dichothorax). Species in this clade have been informally classified as members of the hispidus-group sensu Mackay [29] (T. sp. nr. peninsularis), the arboreal mainland Meso-American 'Macromischa' species of the sallei-species group (T. aztecus, T. salvini and T. fuscatus), and the primarily ground-nesting Meso-American and Caribbean pulchellus-group sensu Baroni Urbani [18] (T. augusti, T. subditivus, T. terricola and T. torrei).

obturator-clade
The obturator-clade is a small group encompassing the obturator-and emmae-groups sensu Mackay [29], and the creolus-group sensu Baroni Urbani [18]. All known species from this group are arboreal, nesting in dead twigs or galls on live trees. Additional undescribed species were recently collected from Puerto Rico and Meso-American cloud forest. The obturator-and salvini-clades are both broadly distributed, having ranges like that of the purpuratus-clade, with members in the southern Nearctic and the Neotropics, including the Greater Antilles.

Palearctic-clade
The Palearctic-clade, as implied by the moniker, is mostly confined to the Palearctic biogeographic realm, with two notable exceptions: the dulotic eastern Nearctic species T. americanus (formerly Protomognathus) and a group that contains many of its hosts (the remainder of the polyphyletic longispinosus and schaumii-groups, which will be referred to as the longispinosus-group below) are nested within this clade; additionally, the Afrotropical T. mpala is nested within the southern Palearctic laurae-species group (sensu Prebus [40]). The socially parasitic corsicus-(formerly genus Myrmoxenus) and muellerianus-(formerly genus Chalepoxenus) species groups are nested within the Palearctic-clade as well, and are sister to [T. americanus + longispinosus-group] and the laurae-species group, respectively. The Palearctic clade is composed of four subclades and is generally, with a few exceptions, ground nesting.
The first subgroup contains the central Palearctic