Introduction
Megachile Latreille [1] is a large, worldwide genus of approximately 1,500 species of largely leafcutting, solitary bees. In the Western Hemisphere they inhabit temperate, arid, and tropical regions extending from Alaska to Tierra del Fuego [2]. There are 118 species native to North America [2]. The abundance of megachilids in California is not surprising given the wide diversity of habitats and microclimates [3], [4].
Leafcutter bees are named for their use of leaf pieces in nest building. They constitute Megachile belonging to Michener’s Group 1 [5] in which bees frequently construct two or more cells in a linear series. Their nesting sites are found under the bark of dead trees, in stems, in the burrows of wood-boring insects or in burrows self-dug in loose soil or those made by other animals [2], [5], [6]. The females use their sharp, serrated, scissor-like mandibles to cut oblong and circular leaf pieces, most likely from plant sources near the nest [2], [5]. They line the nest cavities with overlapping layers of the oblong-shaped leaf disks. The leaf edges are compressed to extrude sap that, in combination with saliva, creates a glue-like substance that keeps the cells sealed and intact [2]. Each cell is provisioned with pollen and nectar by the female before she deposits a single egg on the food mass. After depositing the egg, she seals the cell with one to several circular leaf “caps” [2], [5], [6]. After a few weeks, depending upon species, the eggs hatch, and the larvae develop through multiple instars and feed on the provisions. Mature larvae spin cocoons of two or more layers of silk and diapause as prepupae. Cocoons are sturdy structures [7] made increasingly airtight by the larva’s secretion of a brown liquid that fills and hardens the interstices of the silk layers [6]. This application binds the silk mesh and makes the cocoon extremely durable. Simultaneously, this fastens the cocoon’s outer surface to the surrounding leaf disks that firmly hold the structure together. The larvae subsequently pupate and emerge as adults by chewing their way out through the cap.
That females may spend the majority of their time collecting pollen and nectar to provision their young [2] and construct intricate nests with specific materials indicates a very complex and highly evolved plant-insect interaction, and strongly suggests a long evolutionary history [8]. The use of leaf disks of various sizes, shapes, and textures also reflects highly complex and evolved behavior [2], [8], [9], [10].
As currently known, the megachilid fossil record is restricted to the Cenozoic based on body fossils preserved as compressions and three-dimensionally preserved in amber, as well as trace evidence from fossil angiosperm leaves whose margins show smooth-edged oblong and circular cutouts [8], [11], [12]. Engel [13]–[15] and Engel and Perkovsky [11] have compiled the evolutionary history and an overview of the body fossil record, respectively. Morphological data (body fossils and leaf cutouts attributed to Megachile) and molecular data do not always agree on the time divergence of the genus [8], [11]–[14], [16]–[22]. Although the phylogenetic relationships and evolutionary history of the genus have become clarified as more studies incorporate molecular data [23], the fossil record remains incomplete and some specimens assigned to Megachilidae may need revision. For example, molecular data suggest that Megachilidae arose in the Cretaceous about 140–100 mya, but the genus Megachile is estimated to have originated only 22 mya [23]. However, leafcutters are derived species of Megachile and therefore, the fossil record based on leaf cutouts from the Early to middle Eocene in North American and Europe [16]–[20] suggests that basal divergences in the Megachilini occurred earlier in the Paleocene or Latest Cretaceous [11].
Here we report on fossil Megachile nest cells with pupae (LACMRLP 388E) recovered from the Late Pleistocene Rancho La Brea Tar Pits in southern California. Though geologically young, this is the first report of three-dimensionally preserved Megachile nest cells that shows rare preservation and life-stages. The pupal morphology, nesting behavior, and cell construction of LACMRLP 388E best match M. gentilis, a member of the native Nearctic Litomegachile Mitchell [24], a subgenus that today ranges from southern Canada to Cuba and southern Mexico [5], [25], [26].
The discovery of LACMRLP 388E provides valuable information for better understanding the environmental conditions of southern California in the Late Pleistocene. By setting specimens within a geological as well as an ecological context, Quaternary fossils are shown to be valuable precursors to modern biota [27]. Although the asphaltic deposits at Rancho La Brea are most often associated with vertebrate remains from saber-toothed cats and mammoths, the insects and plants found there are also significant fossils because they are original material, and thus, intact, three-dimensional, and structurally complex. As such they can provide the most valuable paleoenvironmental information for the richest Ice Age fossil locality. Our goal was to synchronize data by identifying both nest cell insect and plant material in order to make the significant paleoenvironmental inferences possible. These efforts resulted in environmental data, that a mesic environment occurred at a lower elevation than today at Rancho La Brea, the well-established provenance for LACMRLP 388E. This research also resulted in new information on M. gentilis, including diagnostic features of its nest cell architecture and insight regarding its relatively conserved ecological niche since the Late Pleistocene.
In additional to its role as a sensitive paleoenvironemental indicator and providing new information on M. gentilis, LACMRLP 388E is of rare value because its remarkable preservation, especially of the intact pupae, is of a standard unusual even for fossil material from Rancho La Brea.
Discussion
The provenance of specimens recovered from Rancho La Brea cannot always be discerned because alluvial wash may have deposited some of the recovered material at a distance from their original location [38]. Nevertheless, the remarkable preservation and lack of water damage to LACMRLP 388E indicates with near certainty that it was assembled in the ground on the site of Pit 91 where it was found. The provenance of the specimen, in combination with environmental niche models and morphometric investigations, constitutes strong and comprehensive evidence to support confident fossil identifications and subsequent paleoenvironmental inferences for the Late Pleistocene, an epoch of particular climatic variation.
Because the leaves used in nest construction were collected in close proximity to each other and at the site of deposition, we can infer from their identification that a woody and riparian habitat existed at Rancho La Brea ∼23,000–40,000 radiocarbon years BP. This inference is supported by Maxent habitat suitability models which suggest that M. gentilis was distributed in a mesic environment that likely occurred at a lower elevation during the Last Glacial Maximum (LGM) ∼21,000 radiocarbon years BP (Fig. S1, Fig. S2). Presently, M. gentilis occurs in higher elevation habitats that constitute the perimeter of its projected habitat suitability during the LGM (Fig. 4). These areas surrounds the Los Angeles Basin in which Rancho La Brea is situated (Fig. 4).
Fossil plants from Pit 91 excavations have been grouped into four categories including a riparian association which includes plants from flood plain and canyon habitats [39]. Aquatic and moisture-loving plants indicate a permanent water source near Rancho La Brea [39], [40]. But additional plants from Pit 91 indicate other associations such as coastal sage scrub, chaparral, and deep canyon [39]–[41]. Most of the excavated plants probably inhabited Rancho La Brea, but others may have been transported by streams or floods [39], [41]. LACMRLP 388E suggests for the immediate Rancho La Brea area the presence of a gallery forest within a riparian zone. These forests exist along watercourses often in arid regions and standout in contrast to e.g. an adjoining grassland or open woodland habitat. The indication of a mesic environment at Rancho La Brea is relatively consistent with coastal run-off records [42] which indicate increased precipitation, possibly associated with increased glaciation [43], at ∼25,000–20,000 years ago. Relative comparison of the geographic distribution of habitat suitability between the contemporary and LGM models reveals that M. gentilis has remained virtually unique to California and southwestern Arizona (Fig. 5) where the average temperature of the coldest quarter rarely falls below freezing (0°C). The contemporary distribution of M. gentilis is limited to mesic environments where the difference between the maximum of the warmest month and the minimum of the coldest month is centered around 33°C (Temperature Annual Range, Fig. S1). That M. gentilis has retained its abiotic niche since the LGM provides a strong benchmark with which to compare the climate restrictions of other fossils from Rancho La Brea.
Most animals and plants excavated from Rancho La Brea are extant, and those that have become extinct are mostly mammals such as common, larger carnivores (e.g. saber-toothed cats and dire wolves), common, medium-to-large herbivores (e.g. the western horse, mastodonts and mammoths), and some birds [39]. Only two species of insects recovered from Rancho La Brea, scarabs that relied on dung from mammals that became extinct, may have died out as well [39], [44]. The current geographic distribution of most of the insect species from Rancho La Brea occurs in warmer parts of the United States and Mexico. Insect damage to the fossil bones attributed to tenebrionid and dermestid beetles is consistent with a warm period of at least 4–5 months at the time that the fossils were trapped [45]. This differs from interpretations of Late Pleistocene climatic conditions in southern California based on pollen from deep sea cores which indicate a shift to a cooler environment, as indicated by a transition from predominantly hardwood scrub-oak vegetation to coniferous pines and juniper [46]–[48] during the Last Glacial Interval from 24,000–14,000 years BP [46]. However, offshore palynological records may not reveal short-term increases in temperature that would result in both the colonization of insects with warm climate restrictions for brief periods due to their higher mobility, and in increased asphalt entrapment from more active seeps [45]. The current geographic distribution and climate restrictions of insect species preserved at Rancho La Brea, along with the physical properties of natural asphalt, indicate that the fossil insects represent warmer intervals of the Late Pleistocene [45]. But this does not resolve the discrepancy that some fossils recovered from Rancho La Brea such as M. gentilis inhabit mesic environments, while most of the tenebrionid beetles preserved at Rancho La Brea inhabit dry scrub and woodland habitats [49]. Further research on insects that can be dated to a specific time period, and that also have a clear provenance for Rancho La Brea, as well as narrow climate restrictions, will greatly enrich our understanding of the paleoenvironment, as well as climate variation during the Late Pleistocene in southern California.
Aside from providing paleoenvironmental data, LACMRLP 388E enhances the fossil record of megachilids and the genus Megachile by providing the first reported specimen from the Pleistocene. This contributes to a better understanding of a genus whose phylogenetic relationships are still being researched.
The examination of LACMRLP 388E also presents neontologists with important information. O’Toole and Raw [2] state that, despite the diversity of nest sites and habitats exploited by leafcutter bees, their nest cells “differ little in structure and nesting behavior,” a trait that makes the cells often difficult to identify. Yet the nest cell morphology of LACMRLP 388E provides one of the most important diagnostic tools to species-level. Nest cell construction of leafcutter bees is rarely closely examined or recorded. Where this has occurred [9], [10], [31], species-specific aspects of nest cell architecture have been documented. We surmise that there is indeed great morphological variation among Megachile species as has been demonstrated for another megachilid genus, Osmia [50]. A record of species-specific materials and nest structure may provide diagnostic tools useful in the field and for an organism that is often difficult to identify at an immature stage.
Materials and Methods
Excavation and Preparation
The cells, which were nested in chunky asphalt-impregnated matrix, were easier to excavate than if they had been preserved in more asphalt-concentrated matrix and therefore, were not exposed to solvent used in other preparation techniques. In addition, the immediate coating of the specimens with glyptol, an alkyd resin, upon discovery may have prevented further breakage during handling and preparation (Obermayr, Pit 91 field notes p. 1770, 1970).
Micro-CT Scanning and Reconstruction
A 3D model was built for each pupae using Micro-CT data (Videos S1-6). Specimens were scanned on a SCANCO Micro-CT Scanner Model V1.2a. Linear Attenuation was 1/cm, 7.000000e+01 kVp with a slice thickness of 5 microns. Micro-CT slices were analyzed and 3D reconstructions of each bee built using Amira 5.4. LACMRLP 388Ea (male) was output as 2172 slices and reconstructed using 3,021,012 triangles (Videos S3, 5). LACMRLP 388Eb (female) was output as 2849 slices and reconstructed using 4,380,432 triangles (Videos S4, 6). To build 3D models of the nests, we scanned each nest on a MicroCAT II small animal CT system (Siemens Preclinical Solutions, Knoxville, TN, USA). Exposure settings were 70 kVp, 500 mAs, with a slice thickness of 2 mm and output as 436 slices. The female nest was built and output at 18,540 triangles. The male nest was built and output at 11,012 triangles. All models were cleaned and smoothed in Geomagic 10.
Use of Comparative Material
The fossil specimens were also compared to material from the comprehensive collection at the USDA-ARS Pollinating Insect Research Lab. The plant materials used and specific architecture was examined from the nest cells of other Megachile species of similar, diminutive size that include western United States distribution.
Estimation of Habitat Suitability at Present and During the LGM
Environmental space use
To investigate differences in environmental space use between M. gentilis and M. onobrychidis, we performed principal components analysis of bioclimatic variables, altitude, and Cartesian coordinates (i.e., decimal degrees latitude and longitude) associated with distribution records. Contemporary environmental space use was estimated from recent specimen locality records (Dataset S1; National Pollinating Insect Database, Logan, UT) for eight informative bioclimatic variables: mean temperature of warmest quarter, mean temperature of coldest quarter, mean temperature of wettest quarter, mean temperature of driest quarter, precipitation of wettest quarter, precipitation of driest quarter, precipitation of warmest quarter, temperature annual range (Fig. S1; [51], http://worldclim.org). The contemporary (∼1,950–2,000 AD) bioclimatic variables were selected based on their ability to capture seasonal trends and species-specific differences between M. gentilis and M. onobrychidis. Differences in the distribution of the study species for each bioclimatic variable were tested with the Wilcoxon rank sum test with significance (P) based on an alpha of 0.95 and visualized with violin plots (Fig. S1). Data concentration ellipses were estimated using the k-means clustering method (K = 2) to visualize contemporary record assignment, as well as assign the environmental space of the La Brea Tar Pits during the LGM to either M. gentilis or M. onobrychidis.
Geographic distribution of habitat suitability
To estimate the geographic distribution of habitat suitability in the western U.S. at present and during the LGM (∼21,000 y BP), habitat suitability models (HSM) were constructed under the principle of maximum entropy with MaxEnt v3.3.1 [52]. Each species’ HSM was constructed from the specimen locality records and bioclimatic variables selected for the principal component and cluster analysis (see Environmental Space Use). Under the principle of niche conservatism [53], we estimated the geographic distribution of habitat suitability of both species during the LGM with analog bioclimatic variables of their contemporary bioclimatic niche. To provide a robust estimate of LGM habitat suitability, we allowed for the HSM to exceed contemporary bioclimatic extremes in the event that maximum entropy was achieved during model construction. Constructing LGM HSMs that are younger than the fossil records of the study species guarantees that both M. gentilis and M. onobrychidis, or a common Litomegachile ancestor were already in existence. The spatial resolution of all bioclimatic variables used in the models is 2.5 arc-minutes. Data processing and geographic visualization of the HSM were implemented in ArcGIS 10.1 (ESRI, Redlands, CA). Model performance was evaluated in MaxEnt using the area under curve (AUC) statistic and a jackknife of variable importance (Table S1). The AUC is constrained between zero and one where values closer to one suggest better model performance. Models were averaged over 50 replicates, with 10-fold cross validation for each replicate for model validation.
Statistical analysis
Outside of MaxEnt, all analyses were conducted in R v3.3.0 [54]. Principal components were visualized with the scatterplot function in the car library. The k-means clustering method was performed with the kmeans function in the cluster library. Violin plots were visualized with the vioplot library.