Habitat Specificity, Host Plants and Areas of Endemism for the Genera-Group Blepharida s.l. in the Afrotropical Region (Coleoptera, Chrysomelidae, Galerucinae, Alticini)

Simple Summary Knowledge of the processes that generate biodiversity is a core-issue of any conservation strategy because it allows predicting the effects of environmental changes in the number and distribution of target taxa. Some phytophagous insects can be good potential indicators of such processes, thanks to their wide distribution and their sensitivity to climate change, due to the association with specific environments and host plants. Unfortunately, this ecological information is often lacking. However, statistical tools allow reconstructing the ecological features of interest, based on the presence–absence data of the taxa, the climatic and vegetational features of their distributional areas, and the available data about their host plants. In this paper, we apply some geostatistical methods to identify processes and patterns of biodiversity at a continental scale, focusing on a group of phytophagous insects widespread in sub-Saharan Africa. Abstract The genus Calotheca Heyden (Chrysomelidae) is mainly distributed in the eastern and southern parts of sub-Saharan Africa, with some extensions northward, while Blepharidina Bechyné occurs in the intertropical zone of Africa, with two subgenera, Blepharidina s. str. and Blepharidina (Afroblepharida) Biondi and D’Alessandro. These genera show different ecological preferences. Through an up-to-date presence–absence dataset, in the light of the terrestrial ecoregions of sub-Saharan Africa and the distribution of their possible host plants, we interpreted the pattern of occurrence of these three supraspecific taxa, by geostatistical analyses in GIS and R environments. The separation of Blepharidina from Calotheca was probably driven by changes in climate as adaptation to more xeric and warm environments with a major occupancy of semidesert and savannah habitats, especially in the Afroblepharida species. Based on our data and analyses, Calotheca is mainly associated with Searsia (Anacardiaceae), and Blepharidina is likely associated with Commiphora (Burseraceae). This hypothesis is also corroborated by the widespread and even dominance of the Commiphora plants in the ecoregions where both Blepharidina s.str. and, above all, Afroblepharida, are more common. The main areas of endemism of the two genera are also differently located: Calotheca in the temperate zone; Blepharidina within the intertropical belt.


Introduction
The Alticini are a tribe of Coleoptera Chrysomelidae comprising over 534 genera and about 8000 species [1,2], occurring all over the world. Members of this tribe are commonly defined as "flea beetles" because of the presence of a metafemoral extensor tendon that enables them to jump [3][4][5]. Adult and larval stages mainly feed on stems, leaves, or roots, although rarely on flowers, of almost all the higher plant families, generally with high levels of specialization and in different environments [6][7][8]. The highest species richness occurs in the tropics of the southern hemisphere, even though our knowledge about the Alticini is still incomplete for these areas [2,[9][10][11].
For a long time, Blepharida Chevrolat, 1836 [12] has been considered a widespread flea beetle genus occurring in the Nearctic, Neotropical, and Afrotropical regions and, to a small extent, in the Palearctic region, with the species C. sacra reported in Israel [10,13,14]. Recently, the species from sub-Saharan Africa, Asia Minor, and the Arabian Peninsula were attributed to the genera Calotheca Heyden, 1887 [15] and Blepharidina Bechyné, 1968 [16][17][18], confining the distribution of the true Blepharida to the Nearctic and Neotropical regions.
Calotheca shows a geographic distribution that includes the greater part of sub-Saharan Africa, with extensions in Israel and Saudi Arabia (Figure 1). Although it is absent in the north-western areas, it is particularly common in the eastern and southern parts of its distribution range [17]. Thirty-two species are currently attributed to this genus [17,19,20]. Blepharidina comprises two subgenera, Blepharidina s. str., hereafter Blepharidina, and Blepharidina (Afroblepharida) Biondi and D'Alessandro, 2017, hereafter Afroblepharida [17]. This genus occurs in the intertropical area of Africa with at least seventeen species of Blepharidina, mostly distributed in the south, and twelve species of Afroblepharida occurring largely in the central eastern area, including the Socotra island, with some species towards the north and one in the west [18,21,22]. The ranges of these two subgenera show an overlap in southern Kenya [8,17] (Figure 1).
Regarding the life cycle, general information is mainly available for Blepharida s.l. [23,24]. Generally, adult females lay eggs on their host stems during the summer; larvae are ectophytic and feed for approximately 20 days, then they eventually drop off the plant and find new shelter in the soil, where they pass through their pre-pupal and pupal stages. At the end of this phase, the adult emerges around the end of spring [25]. Molecular studies have confirmed that Blepharida s.l. species and their host plants, mainly represented by Anacardiaceae and Burseraceae, evolved their traits in response to mutual selective pressures [26].
The ecology of the Afrotropical Afroblepharida, Blepharidina, and Calotheca is very poorly known, including few with definitive host associations. The available records of Calotheca host plants identify the genus Searsia (generally reported as Rhus) from the Anacardiaceae family as primary host plants [23,27]. In this regard, it is important to clarify that the genus Rhus L. has traditionally been defined to include up 250 species worldwide. However, most of the African members of Rhus are now widely recognized to belong in the segregate genus Searsia F.A. Barkley, which is distributed mainly in southern Africa, as well as in Sicily and the Middle-East to Yunnan, with over 100 species [28][29][30]. Other genera of Anarcadiaceae, such as Ozoroa Delile and Schinus L., and the genus Commiphora Jacq. from the Burseraceae, are generically recorded as host plants for Blepharidini in Africa [31,32].
We tried to understand if and how climatic conditions have influenced the current distributions of Afroblepharida, Blepharidina and Calotheca in the Afrotropical region. We used ecological niche modelling, a technique applied for climate-related issues to many other case studies on Chrysomelidae [33][34][35][36], to highlight possible differences in their habitat preferences [8]. Our analysis focused on the climatic conditions, particularly those related to temperature and precipitation patterns, because of the significant influence of these parameters on the different types of vegetation and, consequently, on the distribution of these phytophagous beetles. Our models suggested modifications during the time of the suitable areas of Afroblepharida, Blepharidina and Calotheca led by changes in climate; specifically, the increase in xeric and warm environmental conditions.
Starting from this information, in this research we aim at: (i) interpreting the pattern of occurrence for all known species of Afroblepharida, Blepharidina and Calotheca, through a reliable and up-to-date presence-absence dataset, in the light of the terrestrial ecoregions reported for sub-Saharan Africa [37] and distribution of their possible host plants; (ii) correlating the distribution of Calotheca with the distribution of the Searsia plant genus; (iii) hypothesizing the possible host plants for Afroblepharida and Blepharidina using geostatistical analyses; and (iv) identifying and delimiting eventual areas of endemism.

Study Area and Flea Beetle and Plant Datasets
The study area includes sub-Saharan Africa, Madagascar, and the Arabian Peninsula and its adjacent northern areas representing the northern limit of the distribution of the taxa considered here (Figure 1). The datasets for the flea beetles Afroblepharida (69 occurrence localities), Blepharidina (162 occurrence localities), and Calotheca (797 occurrence localities), for a total of 61 species, were generated using both known localities and original data [17][18][19][20][21][22]. Data refer mainly to material preserved in the world's main museums [17] and private collections and cover over a century. Part of the Calotheca from the Republic of South Africa was collected during expeditions carried out from 1990 to 2010, making it possible to obtain secure information on their host plants. Afroblepharida and Blepharidina, in contrast, have not been the target of recent collecting expeditions, also considering the difficult accessibility because of the political instability of many of the regions included in their distribution area. This is why for the species of Afroblepharida and Blepharidina, the available distributional and ecological data derive exclusively from the study of preserved material [18,21]. The datasets of the plants Searsia (13,605 occurrence localities) with 104 species and Commiphora (6343 occurrence localities) with 142 species, occurring in the African continent and Madagascar, were obtained from the GBIF portal (Commiphora accessed on 27 January 2021 [38]; Searsia accessed on 13 January 2021 [39]) and the available literature and checklists of African countries. The spatial information from the IUCN Red List (https://www.iucnredlist.org) (accessed on 27 January 2021) was also used to compare the occurrence localities such obtained with species and genus range data. All duplicate, doubtful, or low-precision records, as well as data missing some information in their attribute tables, were discarded from the analyses.

Ecoregions
Terrestrial ecoregions of the world (869, last update) were proposed by Olson et al. (2001) [37]. They are relatively large land units containing distinct assemblages of natural communities and species, with boundaries that approximate the original extent of natural communities prior to major land-use change. Ecoregions are classified into 14 major habitats, such as forests, grasslands, or deserts, and represent a useful framework for conducting biogeographical or macroecological research, for identifying areas of outstanding biodiversity and conservation priority, for assessing the representation and gaps in conservation efforts worldwide, and for communicating the global distribution of natural communities on earth. The shapefile used by us, including 132 ecoregions for Africa, Madagascar, and Arabian Peninsula ( Figure S1), was downloaded from http://www.fao.org/land-water/land/ land-governance/land-resources-planning-toolbox/category/details/en/c/1036295/ (accessed on 27 January 2021).

Density of Species Richness and Occurrence
Occurrence localities for each target taxa were processed through the Point Density tool in ArcMap 10.0 [40], to obtain the magnitude of the occurrence density surface based on the neighborhood of localities. Additionally, the Calculate Richness and Endemicity process (tessellation resolution = 100 km) from the SDMtoolbox 2.4 ArcMap toolbox [41] was applied to the occurrence localities of both target species and plants, taking the species as the selected variable to perform the analysis. To obtain continuous responses of Species Richness over the study area, the outputs of the tool were first converted to point features, and then processed through the Kernel Density tool (output cell size = 1 km) in ArcMap 10.0 [40].

Habitat Specificity
To determine the magnitude of a patch contributing to the overall richness at a vast scale, we applied the Habitat Specificity index [42] to the Afroblepharida, Blepharidina and Calotheca occurrences as a source of species' information, using the ecoregions reported for sub-Saharan Africa [37] as target landscape patches. Considering that the latter are shaped following ecological boundaries (thus, in a non-standardized pattern), we excluded the "area" contribution of the formula by adopting the Halvorsen and Edvardsen correction [43], as suggested [42,43] and applied [44] in other cases. The corrected Habitat Specificity (S) of a patch (in this case, a single ecoregion) was thus calculated as Sij = ∑ (mj /ni), with m representing the number of species occurring in the jth patch, and n being the number of patches in which the ith species occurs, referring to the total landscape.

Flea Beetle-Plant Species Associations
To assess the possible associations of Afroblepharida and Blepharidina with Commiphora plants, as well as to confirm those existing between Calotheca with Searsia plants, we calculated the spatial correlation between their respective kernel density rasters for Species Richness (obtained through the SDMtoolbox software, see above). The Band Collection Statistics tool in ArcMap 10.0 [40] was used for this aim. Moreover, 19 climatic variables were downloaded from the Worldclim.org repository ver. 2.1, at 30 arc-seconds spatial resolution [45]. Their averages (Table 1), calculated for all occurrences for every taxa, were used to carry out a cluster analysis (Euclidean distance and WPGMA clustering method) among Afroblepharida, Blepharidina, and Calotheca flea beetles, and Searsia and Commiphora plants, to evaluate the possible associations between the insects and the relative host plants. Statistical analyses were performed using NCSS11 software (NCSS, LLC, Kaysville, UT, USA). Finally, to detect the possibility of a niche overlap driven by climate, we evaluated the target flea beetle and plant occurrences by using the "hyperoverlap" package [46] in R environment [47]. This tool permits the evaluation of overlap or divergence between point datasets, such as in our case, using support vector machines to find the best classification (linear or polynomial), giving a classification matrix, for any n-dimensional set of point attributes [46]. The "hyperovelap_detect" function, which has proven to be resistant to sampling biases and to small numbers of points [46], was used to find climatic niche overlap for the aforementioned pairs, while the "hyperovelap_lda" function was used to obtain the three-dimensional plots (reporting a combined linear discriminant analysis, PCA 1 and PCA 2 residuals) resulting from the classification. This last function was needed because four of the Worldclim climatic variables were used for this analysis, namely, BIO1 (mean annual temperature), BIO7 (temperature annual range), BIO14 (precipitation of the driest month) and BIO18 (precipitation of the warmest quarter), identified to be the most relevant variables from the species distribution models built by D'Alessandro et al. [8], shared among Afroblepharida, Blepharidina and Calotheca.

Areas of Endemism
To identify the areas of endemism for Afroblepharida, Blepharidina, and Calotheca, we used the Geographical Interpolation of Endemism (GIE) toolbox [48] for ArcMap 10.0. This method is independent of grid cells and is based on estimating the overlap between the distribution of species through a kernel spatial interpolation of centroids of species distribution and areas of influence. The latter is defined as the distance between the centroid and the farthest point of occurrence of each species. In this study, two categories of endemic species were considered: species with up to 100 km (class 1) and 300 km (class 2) of distance between the centroid and the farthest point. Only areas with at least two synendemic species were generally considered.

Habitat Specificity and Flea Beetle-Plant Species Associations
The application of the Habitat specificity index identified the most important ecoregions that characterize the distribution of Afroblepharida (Figure 2), Blepharidina ( Figure 3) and Calotheca (Figure 4) in terms of the magnitude that each ecoregion had in contributing to the overall richness. Afroblepharida, with its 12 species, clearly shows more xeric preferences. It occupies 13 different ecoregions, with over 75% of the area occupied by the "Northern Acacia-Commiphora bushlands and thickets" (31.2%), "Somali Acacia-Commiphora bushlands and thickets" (31.2%), and "Sahelian Acacia savanna" (14.4%) ecoregions. For the 17 species of Blepharidina, 20 ecoregions were identified, but over 60% of the total area is represented by the "Northern Acacia-Commiphora bushlands and thickets" (30.9%), "Angolan Miombo woodlands" (15.2%), and "Western Congolian forest-savanna mosaic "(15.2%) ecoregions. The coverage in ecoregions resulting for the 32 species of Calotheca was distinctly different. It includes 46 ecoregions, and about 42% of its extension is represented by only two ecoregions named "Drakensberg montane grasslands, woodlands and forests" (24.0%) and "Southern Africa bushveld" (17.9%).
The results of raster correlation between the point densities ( Figures 5 and 6), obtained for the occurrence localities for each taxon, gave a high correlation (Pearson's r) for the Calotheca-Searsia pair (r = 0.676), as well as for the Afroblepharida-Commiphora (r = 0.576) and Blepharidina-Commiphora (r = 0.594) pairs. On the other side, the opposite pairs       Beyond the magnitude that each ecoregion had in contributing to the overall richness, the climatic niche resulted as overlapping for Afroblepharida-Commiphora, Blepharidina-Commiphora and Calotheca-Searsia pairs ( Figure 8); the misclassified points (i.e., the points which could not be assigned to one taxon only of each pair, because of the niche overlap) for the target flea beetles were 66.7% for Afroblepharida-Commiphora (number of support vector machines = 71), 38.7% for Blepharidina-Commiphora (number of support vector machines = 208), and 99.9% for Calotheca-Searsia (number of support vector machines = 1369) pairs. Instead, no overlaps were found for the Afroblepharida-Searsia, Blepharidina-Searsia and Calotheca-Commiphora pairs.

Areas of Endemism
The consensus map of the main areas of endemism identified by GIE and generally characterized by at least two synendemic co-generic (Calotheca) or co-subgeneric (Afroblepharida and Blepharidina s.str.) species is reported in Figure 9. For Afroblepharida, two main areas were identified in intertropical Africa: the first, with four synendemic species

Discussion
The Blepharida genera complex (Blepharida s.l.) has a possible Gondwanan origin, with the separation of the close Malagasy genus Xanthophysca Fairmaire consequent to the break-up between Madagascar and eastern Africa (approximately 165-160 Ma), and of the genus Calotheca consequent to the separation between South America and Africa (approximately 130 Ma), confining the genus Blepharida Chevrolat s.str. in the American continent only [8,13,17,26,31,49]. No insights are currently available about the time origins of Blepharidina and Afroblepharida. Their separation from Calotheca in Africa was probably driven by changes in climate as adaptation to more xeric and warm environmental conditions, with a major occupancy of semidesert and savannah habitats [8]. This led to the current partially parapatric scenario, in which the suitable areas of Calotheca instead show a significant major occupancy of grasslands, shrublands, and forest habitats. Even the main areas of endemism identified for the three taxa show significant differences in their geographical location. Those of Calotheca are in two temperate zones (Drakensberg and Western Cape Province), while those of Blepharidina (southern Kenya/north-eastern Tanzania and north-western Angola/south-eastern Democratic Republic of Congo) and Afroblepharida (eastern Ethiopia/central Somalia and central/southern Kenya) are within the intertropical belt, with a wide overlap observed in southern Kenya.
The overall results of geostatistical analyses, whose applications are proven to give reliable insights about biodiversity-related issues [34,35,50], resulted in suggesting that both abiotic and biotic factors influenced the distribution of the target species and their co-occurrence. In fact, although abiotic factors seem to significantly affect and explain the distribution patterns of Afroblepharida, Blepharidina and Calotheca, the distribution of their host plants probably played an important role too [8]. Through its evolution, Blepharida s.l. has maintained a close relationship with its host plants, Anacardiaceae and Burseraceae [25,31]. These two plant families represent a pantropical sister pair, which have identical ages (origins in the Cretaceous age) and approximately the same number of species distributed on every continent except Antarctica. Unlike Anacardiaceae, which have shifted their climatic niches frequently during the past 100 million years, including the colonization of the temperate biomes, Burseraceae experienced fewer climatic niche expansions. In fact, there are no frost-tolerant species, and all Burseraceae are restricted to tropical and subtropical latitudes. Some of the lineage divergences in these two plant families may have been due to vicariance, but it is more probable that the great majority of them have been due to long-distance dispersal events and that both Anacardiaceae and Burseraceae have moved easily across the oceans [51].
The New World species of Blepharida mainly feed on Burseraceae and, to a lesser extent, on Anacardiaceae [13]. However, in Africa, Calotheca species are mainly associated with Anacardiaceae of the genus Searsia, and based on our analyses, the host plants of Afroblepharida and Blepharidina species are very probably Burseraceae of the genus Commiphora. This hypothesis is also well supported, beyond all our analyses, by the fact that the Commiphora species are common and even dominant in the ecoregions in which Blepharidina and, above all, Afroblepharida, are more present, such as "Northern Acacia-Commiphora bushlands and thickets" and "Somali Acacia-Commiphora bushlands and thickets".

Conclusions
Our research reported that a multiple-scale and -predictor approach can give very useful clues for the distribution patterns of species which are possibly linked by an ecological relationship. The application of landscape-based methods, such as the Habitat specificity index, coupled with GIS techniques implemented through occurrence and species' geographic information, resulted in a multifaceted pattern of results, showing, from different angles, the plant-host relationship we hypothesized. Additionally, the support offered by multivariate statistics and machine learning methods enabled the incorporation of additional predictors, thus encompassing both the biotic and abiotic components into our general framework of interpretation of the biogeography and ecology of the target taxa.

Data Availability Statement:
The datasets for the flea beetles Afroblepharida, Blepharidina, and Calotheca, generated using both known localities and original data, can be requested from the authors. The datasets of the plants Searsia and Commiphora, obtained from the GBIF portal (Commiphora accessed on 27 January 2021 [38]; Searsia accessed on 13 January 2021 [39]) and the available literature and checklists of African countries, can also be requested from the authors. The ecoregion shapefile was downloaded from http://www.fao.org/land-water/land/land-governance/land-resourcesplanning-toolbox/category/details/en/c/1036295/ (accessed on 27 January 2021).