Tanopicobia gen. nov., a new genus of quill mites, its phylogenetic placement in the subfamily Picobiinae (Acariformes: Syringophilidae) and picobiine relationships with avian hosts

A new monotypic genus Tanopicobia gen. nov. is established for a new species Tanopicobia trachyphoni sp. nov., parasitizing Trachyphonus erythrocephalus Cabanis, 1878 (Piciformes: Lybiidae) from Tanzania. In phylogenetic analyses based on morphological data and constructed using the maximum parsimony approach, this taxon falls within the subfamily Picobiinae Johnston and Kethley, 1973 in the Neopicobia-species-group as closely related to the genus Pipicobia Glowska and Schmidt, 2014. Tanopicobia differs from Pipicobia by the following features in females: genital setae absent; setae ve are situated far and posteromedial to the level of setal bases vi; setae 3a are thick and knobbed. Additionally, a new generic key for subfamily Picobiinae is constructed and general host-parasite ecological and phylogenetic relationships are discussed. Picobiines are present in several lineages of neoavian birds, from basal Galloanseres to terminal Telluraves, which are infested by 70 (89.7% of all) species of these ectoparasites.


Introduction
The subfamily Picobiinae Johnston and Kethley, 1973 (Acariformes: Prostigmata: Cheyletoidea: Syringophilidae) represents taxonomically diverse group of obligate, permanent and highly specialized mite parasites of birds [1], occurring in all zoogeographical regions besides Antarctica [2]. They occupy exclusively short quills (calamus) of the contour feathers, except the enigmatic and monotypic genus Calamincola Casto, 1977 that is found in the quills of wing feathers of neotropical ani cuckoos [1,3,4]. In this microhabitat, they live, reproduce and feed on soft tissue fluids by piercing the quill wall with their long and styletiform movable cheliceral digits. Only young, fertilized females disperse and infest newly developing quills via a natural opening in the quill wall, "superior umbilicus" [5]. All

Material and methods
The mite material used in this study was collected from dry bird skins deposited in the Bavarian State Collection of Zoology, Munich, Germany. Quills of contour feathers were examined using a dissecting microscope and opened with a fine scalpel. Before mounting, all collected mites were softened and cleared in Nesbitt's solution at 60˚C for 12 h [5]. Identifications and drawings of mite specimens were carried out with a ZEISS Axioscope (Carl-Zeiss AG, Germany) light microscope equipped with DIC optics and camera lucida. In the descriptions below, the idiosomal setation follows Grandjean [9] as adapted for Prostigmata by Kethley [10]. Nomenclature of leg setae follows that proposed by Grandjean [11]. Morphological terminology follows Skoracki [5]. All measurements are in micrometers (μm). Measurement ranges for paratypes are given in brackets following the data for a holotype. Common and scientific names of the birds follow Clements et al. [12].

Phylogenetic analysis
Although the main goal of the study was to examine relationships at the generic level, all operational taxonomic units (OTUs) were represented by taxonomic species (morphospecies) in the cladistic analysis. Picobiine genera were represented by one species of each genus (S1 Table). Syringophilus bipectinatus Haller, 1880, representing the second syringophilid subfamily-Syringophilinae Lavoipierre, 1953, was used as an outgroup. Because each particular picobiine genus is represented by a single species in the present analysis (S1 Table), character states appearing as autapomorphies represent true synapomorphies for genera. The autapomorphic characters included in the analysis are essential for generic diagnoses and useful for future phylogenetic studies at lower taxonomic levels [13]. A total of 13 OTUs and 32 morphological characters (among them 15 autapomorphies) were included in our data matrix (S1 and S2 Tables). A detailed discussion of the morphological characters used in the present study is provided by Skoracki [5] and Skoracki et al. [1]. Constructing of the taxa matrix was done using NEXUS Data Editor 0.5.0 [14]. Analyses of character distribution on trees were performed in WINCLADA [15]. Only qualitative, unordered, and a priori unweighted characters were used in analyses. We applied a multistate contingent coding strategy, [16] which is considered as the most useful among available approaches [17]. Following this strategy, characters having multiple states were interpreted as unordered and were not modified into binary characters. In the data matrix, missing states were coded as "?" and inapplicable as "-".
Reconstruction of phylogenetic relationships was performed with PAUP 4.0 beta version for IBM [18] in conjunction with PRAP2 [19] to conduct a ratchet analysis (1000 iterations; 10 random cycles, collapsed zero-branches in effect; options are the default). Nodal support was evaluated by Bremer indices calculated with PRAP2. Analysis of character distributions, drawing, and editing of the trees were performed in TreeView 1.5.2. [20].

Visualization of host phylogeny
To visualize host phylogeny, a tree was constructed based on consensus avian phylogenetic tool available at http://birdtree.org/ [21]. For each bird order, host species of the first picobiinae mite found out in particular host order (see [2]) was used in the analysis. As the source of data, we used the "Hackett All Species tree" with 1000 randomly generated trees. Currently, this tool is widely used in bird evolutionary ecology studies (e.g., [22,23], including the investigation the host phylogenies of bird parasites [7,24]). The most credible tree was then determined using the tool TreeAnnotator v1.8.2 in the software BEAST v1.8.2 [25]. The consensus tree was then graphically adjusted in FigTree v1.4.2 [26].

Bipartite networks and statistics
To visualize pattern in studied parasite-host ecological web, we used a "bipartite" package for R [27]. A number of picobiine mite species infesting each bird order were used as a quantitative indices (Table 1).

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:CB6C15E1-5608-4404-8FEB-3001C8D366A4. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.
Type species. Tanopicobia trachyphoni sp. nov. Host range. Piciformes (Lybiidae). Distribution. Afrotropical region (Tanzania). Differential diagnosis. This new genus is closely related to Pipicobia Glowska and Schmidt, 2014 which representatives are associated with passerines (of the families Acanthizidae, Locustellidae, Monarchidae) and parrots (Psittaculidae) [1,28]. In females of both genera, the hypostomal apex is tapering; the peritremes are mouth-shaped and with ill-visible borders between chambers in lateral branches; the opisthonotal and genital lobes are absent; bases of setae 1a are separated; the apodemes I are with small thorn-like protuberances; solenidia phi (φ) on tibiae I are absent; and physogastric form is with weakly enlarged idiosoma.
Tanopicobia gen. nov. differs from Pipicobia by the following features: in females of Tanopicobia, bases of setae ve are situated far and posteromedial to the level of setal bases vi; setae 3a are thick and ornamented; genital setae are absent. In females of Pipicobia, bases of setae vi and ve are situated in close proximity, and ve are situated postero-or posterolateral to the level of setal bases vi; setae 3a are thin and similar to 1a; genital setae are present.
Etymology. The name of this genus is a compilation of the "Tano"-African river god of war, one of the sons of main Akan deity Nyame, and "Picobia"-type genus for the subfamily Picobiinae.
The clade Picobia-generic-group is monophyletic and split into two clusters: cluster I-(Gunabopicobia+Phipicobia)+Columbiphilus, and cluster II-Picobia+Pseudopicobia. The Neopicobia-generic-group is monophyletic where the genus Tanopicobia is closely related to Pipicobia, with Charadriineopicobia as a sister group.

Discussion
Based on phylogenetic analysis of the family Syringophilidae, the picobiines are placed in the core of the syringophilid tree, and the subfamily Syringophilinae is paraphyletic with respect to this group [29]. They are considerably more morphologically specialized than syringophilines and possess some advanced features like physogastry, which together with K reproductive strategy (few, but large eggs laid by a female [5]) probably allows them to occupy successfully small, but very numerous and always accessible quills of the contour (body) feathers [29]. Thus, picobiines are probably able to effectively avoid competition with other syringophilids and form an evolutionary line parallel to the syringophilines. Although it is possible to find 2-4 species of quill mites on one host, living in different types of feathers [5] and representing a different niches, there are no records of several picobiine species sharing one host; i.e., body feathers are always infested by only one picobiine species.

Questionable placement of the genus Calamincola
The position of the enigmatic genus Calamincola remains unresolved.
In our study, the Calamincola represents either a sister group to all other picobiine genera (Fig 4A) or is a sister group to the genera forming Picobia-generic group (Fig 4B). It is worth to note that monoxenous C. lobatus represents the only species of the picobiine mite able to infest the quills of wing feathers, whereas all other genera occur entirely in contour feathers [1,3,4,24]. It is possible that the wing feathers, i.e., primaries and secondaries, are the ancestral type of syringophilid habitat, as the majority of the family Syringophilidae representatives are associated with the quills of these feathers [29][30][31][32]. Despite mites of the subfamily Picobiinae mostly dwell in the contour feathers, they possibly originally lived in wing quills [29]; thus, the archaic genus Calamincola could represent the "living fossil" among the syringophilid mites,    [21]. Interaction weight depicts a number of picobiine mite species parasitizing particular host order (Table 1). Bird orders included in Telluraves and their relationships with their ectoparasites in green.
https://doi.org/10.1371/journal.pone.0225982.g005 however, this questions needs more in-depth investigation. It is obvious that the future research including molecular data is needed to resolve the position of this genus in the phylogenetic tree of syringophilid mites.
Genus Rafapicobia (11 species) is associated with all three mentioned orders: Passeriformes, Psittaciformes, and Piciformes (Table 1 and Fig 5), thus, with both main clades of Telluraves. Moreover, one species of the genus, R. melzeri Skoracki et al., 2014, was also recorded on five host species of the rather distant family of rails (Gruiformes: Rallidae), which is outside Telluraves. Most probably, it is an example of a horizontal switch of picobiine parasites to phylogenetically distant hosts. To summarize, the host spectrum for Neopicobia-generic group cover advanced bird clades of telluriform birds plus neoavian Charadriiformes and Gruiformes (Table 1 and Fig 5). Despite these two bird taxa comprise well supported subclades in most recent phylogenomic studies [40,47,48], Reddy et al. [39] dubbed them, together with hoatzin (Opistomorphes), "orphaned"orders due to their uncler position within Neoaves. However, both are definitely rather distant from telluravian lineage. For a resolution to which extent host-parasite relationships in Neopicobia-generic group are a result of co-speciation with their host and/or which are host-switches, a more detailed investigation is needed.
For the future, the phylogenies based both on mite morphological and molecular data are needed. Such analyses can be used to group the numerous picobiine (and syringophilid in general) genera described to date and provide a solid basis for the analysis of the co-evolutionary relationships between these parasitic mites and their bird hosts. There are, however, two main problems, which complicate co-evolutionary reconstructions of the picobiine relationship with their hosts. Firstly, subfamily is rather uniform morphologically, as it possesses only a limited set of the external morphological structures [1]. They are represented mostly by setae, where the combination of traits such as the presence/absence of particular setae are the main generic characteristics. Such features have a high probability of being of homoplastic origin and are, therefore, less reliable in phylogenetic analyses (see also [28]). Secondly, material suitable for molecular analyses is absent for most picobiine taxa (which is true also for syringophilids as a whole) as the main body of quill mite species descriptions come from old bird skins in museum collections [54]; therefore, there are certain problems with the ancient DNA isolation from the material [55,56,57]. We are aware of the flaws of our morphological approach, but we believe that our data will be helpful as a background for future molecular-based studies as they can provide useful diagnostic synapomorphies [58].
Supporting information S1 Table. Data matrix of character states for Picobiinae and outgroup taxa. Character states are scored as 0 to 3, inapplicable states as "-", missing states as "?". (DOCX) S2 Table. Characters used in the phylogenetic analysis. (DOCX)