AMERICAN MUSEUM NOVITATES Number 3893, 31 pp. February 22, 2018 Nesting Biologies and Mature Larvae of Oxaeine Bees (Apoidea: Andrenidae) JEROME G. ROZEN, JR.1 ABSTRACT This study encompasses a number of field encounters by the author and others with nests of representatives of three of the four genera of the little-known New World andrenid subfamily Oxaeinae. Species treated include Protoxaea gloriosa Fox, Oxaea flavescens Klug, O. austera Gerstaecker, and Mesoxaea nigerrima (Friese), leaving the nesting biology of only the mono- typic genus Notoxaea completely unknown. Nests, all subterranean, are described and dia¬ grammed, and each is reported to consist of a moderately to very deep main burrow with vertical cells occurring at the lower end attached to the main burrow by subhorizontal lateral tunnels, each of which is closed immediately after egg deposition. To the extent known, eggs, mature larvae, and pupae are described. Two known cleptoparasites of the subfamily are reported: Triepeolus kathrynae Rozen, hosted by P. gloriosa, and Thalestria spinosa (Fabricius) (= T. smaragdina Smith), which attacks nests of both O. flavescens and O. austera. The mature larvae of these cleptoparasitic Nomadinae are described and illustrated as an appendix. INTRODUCTION This paper presents and analyzes the nesting biology of bees of the subfamily Oxaeinae of the Andrenidae. It is based both on (1) fieldwork carried out by the author and associates over the past 47 years when nesting sites of these ground-nesting bees were encountered and (2) accounts in the literature by others. In addition, the paper explores and describes the anatomy of oxaeine last larval instars and other immature stages available as a result of the fieldwork. After a section treating Methods and brief historical listing of biological studies of the subfam- 1 Division of Invertebrate Zoology, American Museum of Natural History. Copyright © American Museum of Natural History 2018 ISSN 0003-0082 2 AMERICAN MUSEUM NOVITATES NO. 3893 FIGURE 1. Nesting site of Protoxaea gloriosa at 1 mile north of Rodeo, New Mexico, August 1970. View of area where numerous nests were discovered and excavated, demonstrating habitat, with K.C. Rozen standing to the left of excavation equipment and the Chiricahua Mountains of Arizona in the background at left. ily, the comparative treatment of known nesting biologies of the subfamily is presented. At the end, an account of available last-stage larvae, eggs, and pupae of the subfamily is offered. Until now little had been reported on this small New World group of fast-flying bees. While this paper fills in a good many details about them, there is still more to learn. The generic classifica¬ tion of Hurd and Linsley (1976) is followed throughout. Methods After a nest entrance is discovered, a hole is excavated a short distance from one side of it with shovel and trowel. The earth is then removed with trowel and penknife from the excava¬ tion toward the descending burrow to expose the path of the burrow in side view. Talcum powder or another dry, white powder can be blown down the burrow with a plastic squeeze bottle to better follow it (fig. 2). As the burrow descends farther, the excavation hole is deep¬ ened as needed, and the accumulation of excavated soil can be removed from the hole if neces¬ sary. Observations and diagrams of the shapes and sizes of nest elements are recorded as field notes. In chipping away the soil close to the burrow or cells with a penknife, a collecting aspira¬ tor with screen removed is used to provide a strong source of wind to blow away obscuring loose soil to reveal nest elements, When lateral tunnels and cells are encountered, records are made of orientation, depth, sizes of cells, cell contents, and ages. Thus a lateral diagram of the 2018 ROZEN: NESTING BIOLOGIES OF OXAEINE BEES 3 FIGURES 2-7. Same area and date as figure 1. 2. Main tunnel of partly excavated nest, approximate side view. White, dry plaster-of-Paris powder marking curved path of open descending tunnel. 3. Excavated nodule containing cluster of four cells (arrows) adhering to one another because of thick walls. 4. Single cell opened perpendicularly to show shape and characteristic shiny, waterproof lining. 5. Cell with upper part removed to view curved egg at center of top surface of provisions, approximate lateral view. 6. Cell with top part removed showing circular wall of cell at midlength with intermediate stage feeding larva on top surface of very moist, orange provisions. 7. More mature larva than in figure 6 with one side of body now submerged while feeding on provisions that have further liquefied. 4 AMERICAN MUSEUM NOVITATES NO. 3893 FIGURES 8-13. Nest components of Oxaeinae in the American Museum of Natural History, photographed at the time of preparation of this manuscript. 8. Unit of cells of Protoxaea gloriosa collected in 1970 demon¬ strating hardness of cell and tunnel walls. 9. Close-up of one opened cell with curved tunnel wall attached, showing smooth water-retardant cell inner surface with dried orange provisions on lower surface. 10. Opened curved tunnel of P. gloriosa, approximate top view, showing series of septa resulting from cell closing and position of cell wall attached below. 11. Plaster cast of open cell and connecting tunnel, demonstrating accu¬ rate shape of cell of P. gloriosa. 12,13. Inner surfaces of spiral closures of two cells of P. gloriosa. 14,15. From nests of Mesoxaea nigerrima, collected from Chamela, Jalisco, Mexico, 1986.14. Cell, side view, demonstrating smooth lining to inner surface and hard cell wall, lateral view. 15. Cell closure, showing spiral closure. nest is established. With more complicated nests where a top view is required, visualization of the cell distribution is made from above before measuring the distance and direction between the burrow and each cell. Samples of nest parts such as cells and entrance tunnels in addition to immature stages are retained for future detailed examination and comparison (fig. 8). Illustrations of immature stages were prepared with a camera lucida attached to a Leitz Wetzlar stereomicroscope. In diagrams of immature stages, outlines indicated by dots are approximations of areas that were missing on the actual specimens. Photographs of anatomical structures were taken with a Cannon Power Shot A2300HD handheld to the ocular of a Leitz 2018 ROZEN: NESTING BIOLOGIES OF OXAEINE BEES 5 Wetzlar stereomicroscope and a Carl Zeiss compound microscope. SEM micrographs were taken using a Hitachi S5700 in the Microscopy and Imaging Facility of the American Museum of Natural History Previous Studies Bertoni (1911) reported finding in-ground nests of Oxaea austera Gerstaecker in Paraguay. The deepest had a vertical burrow that descended about one meter, and some burrows were “curved,” presumably meaning they descended at varying angles. Cockerell (1933) briefly described a massive nesting site of Mesoxaea texana (Friese), as Protoxaea texana. Linsley and Michener (1962) provided an account of a nest of Mesoxaea nigerrima (Friese) (as Protoxaea nigerrima) from Mexico. Details are described below. Truxal (1962) reported finding nests of Oxaea sp. in Peru. Rozen (1964) published the description of a mature (postdefecating) larva of Protoxaea gloriosa Fox based on specimens collected in southwestern New Mexico a year earlier by M.A. Cazier and M.A. Mortenson and given to Rozen for investigation. The paper also included the description of the presumed first instar collected by Cazier, Mortenson, and E.G. Linsley from the same locality. Based on data presented below, that specimen now is almost certainly a second instar. In that 1964 paper Rozen purposely did not provide information concerning nesting biology because he presumed it would be presented by others. Roberts (1973) produced a detailed treatment of the immature stages and biology of two nests of Oxaea flavescens Klug. Rozen and Ricardo Ayala in 1986 undertook a study of nests and immatures of Mesoxaea nigerrima (Friese) at Chamela, Jalisco, Mexico, that has remained unpublished until now. Sar- zetti et al. (2014) provided a detailed account of the nesting biology of Oxaea austera Ger¬ staecker, reporting that nests were occupied by more than one female and were utilized by more than one generation. The current author (J.G.R.) had been unaware of this important study until he received a copy of Ichnoentomology (Genise, 2017) in which it was referenced. This paper supports some of the tentative conclusions on nesting biology of the subfamily that were deduced in the first draff of the current paper, in that O. austera also has nests containing more than one female and these may be used by successive generations. Because of these studies, the following report encompasses representatives of three of the four genera assigned to the subfamily. Only the biology and last larval instar of the South American Notoxaea ferruginea (Friese), the sole known representative of its genus, remains unknown. NESTING BIOLOGY OF THE OXAEINAE Protoxaea: Of the oxaeine nests that have been studied those of Protoxaea gloriosa have been examined most often (see table 1) and in greatest detail. The following account is based on the examination of nests made first by J.G.R. and the late Kenneth C. Rozen in 1970 at one mile east of Rodeo, Hidalgo Co., New Mexico. In 1989 nests were again discovered five miles 6 AMERICAN MUSEUM NOVITATES NO. 3893 TABLE 1. Nest Statistics of Oxaeinae. Numbers in parentheses = number of data when known. Taxon Cell Depth (cm) Burrow Diameter Cell length; diameter Source Oxaea flavescens 195-245 cm 24 mm; 13 mm Roberts, 1973 Oxaea austera 100 cm Bertoni, 1911 90-110 cm 9 mm 2.4 mm (15); 1.2 mm (15) Sarzetti et al., 2014 Oxaea sp. ca. Vi inch Truxal, 1962 Protoxaea gloriosa 24-42 cm 8-10 mm 22-23 mm (6); 10 mm (7) J.G. and K.C. Rozen, field notes, 1970 44-50 cm 7-9 mm (6) 22-25 mm (8); 10-11.8 Rozen and Foster, mm (19) field notes, 1989 36-46 cm 10 mm J.G and B.L. Rozen, field notes, 1990 Mesoxaea texana “several feet” 2 mm Cockerell, 1933 Mesoxaea nigerrima 58 cm 0-12 mm 28-30 mm (2); 13-15 mm Linsley and Michener, (2) 1962 32-58 cm 0.5-11 mm 25-29 mm (7); 12.5-13 Rozen and Ayala, mm (9) field notes, 1986 east of Sahuarita, Pima Co., Arizona, by Robert L. Foster and J.G.R., and in 1990 the late Bar¬ bara L. Rozen and J.G.R. studied them eight miles northeast of Portal, Cochise Co., Arizona. Diagrams of six nests (from a total field of about 17) have been selected from field notes and are reproduced here (figs. 20-24, 26) illustrating the diversity of nest structures. Following the treatment of nests of P. gloriosa, nests of other Oxaeinae are interpreted in light of our present understanding of P. gloriosa. The overall nest pattern of this species in side view (figs. 20-24, 26) consists of a lengthy main tunnel that is mostly open. It descends following a somewhat crooked, irregular path to the cell level usually with an open vertical cell connecting to the end of the open main tunnel by a more or less horizontal open lateral tunnel 8-14 cm long. This cell was the one being provisioned at the time of nest discovery. Other nest cells, all vertical (or nearly so) and closed, occur at about the same level and are presumably connected to the main burrow by soil-filled and, therefore, often obscure laterals. All nest entrances observed were on nearly horizontal ground sometimes next to a rock or clumps of low vegetation. Main tunnels as well as laterals ranged 8-11 mm in diameter with most at 10 mm in diameter. Laterals (figs. 11, 16) narrowed several mm at cell entrances. After closing a cell, the female fills the lateral with soil before excavating the next lateral and cell. Main tunnels remain mostly open although some parts (figs. 23, 26) may be temporarily blocked with a short stretch of soil, presumably for a brief period during cell construction or closure. Some main burrows (figs. 22, 23) revealed a large, irregular enlargement partway down, here termed quarry excavations, almost certainly the result of a female removing soil to backfill a lateral after cell closure following egg deposition. At other times lateral fill is derived from new cell construction. Surface tumuli at burrow entrances are sometimes visible (figs. 20, 23). Although the irregular descending pattern of 2018 ROZEN: NESTING BIOLOGIES OF OXAEINE BEES 7 FIGURES 16-19. Diagrams of cells and egg of Protoxaea gloriosa. 16. Demonstrating exact shape of lumen and entrance tunnel as well as approximation of cell and tunnel wall. 17. Cell with provisions showing position of egg on provisions with subsurface bubbles presumably of gas. 18. Cell with postdefecating larval of P. gloriosa with moldy fecal material applied to upper end. 19. Egg, lateral view, anterior end to left. main burrows may be due to rocks or roots that obstructed the constructing female as she descended, it may also be an adaptation to avoid rain water from reaching open cells since rainwater is likely to be absorbed through unlined burrow walls along the way. Vertical open cells with waterproof linings would be at risk if straight open main tunnels led directly to them. The long lateral tunnels of this species serve the same function as a crooked main tunnel since they too lack waterproof linings. Furthermore, although laterals tend to be subhorizontal, they almost invariably (figs. 20-23) curve upward a short distance in front of the cell and then curve strongly downward to join the top of the vertical cell, thus serving to protect the cell lumen from flooding with water. The curvature allows for further absorption of rainwater and also possibly may provide a “plumber s trap.” Cells of a nest tend to occur at nearly the same depth rather than being distributed along a descending tunnel, as is the case with many bees. Cells in large nests seem to group into small clusters (fig. 26). When diagramed in lateral view they may seem to cluster into larger groups, but when viewed from above they are seen to be the result of long laterals radiating outward in various directions at one depth (figs. 24-27). While small clusters seem to represent the work of a single female because of the ages of the contained offspring, it is unlikely that larger nests represent the activities of a single female. Possibly several females are simultaneously using the same nest entrance or it may be evidence of successive generations. This information is sup- AMERICAN MUSEUM NOVITATES NO. 3893 FIGURES 20-23. Diagrammatic representations of patterns of simple nests of Protoxaea gloriosa with dimen¬ sions and shapes approximated, side view, identified by year of discovery. 20. 1970. With three cells. 21. 1990. With five cells. 22. 1990. With four cells and quarry excavation. 23. 1989. With one cell and quarry excava¬ tions; note short blockage of lateral! 2018 ROZEN: NESTING BIOLOGIES OF OXAEINE BEES 9 FIGURES 24-27. Diagrammatic representations of more complex nests of Protoxaea gloriosa, side and top views of each nest, both discovered in 1970. 24, 25. Demonstrating complex distribution of immature stages in cells, with sequence of egg deposition indicated by approximate age of immatures: cell 1, unknown; cell 2, one-half grown; cell 3, two-thirds grown; cell 4, unknown; cell 5, four-fifths grown; cell 6, three-fourths grown; cell 7, one-fifth grown; cell 8, full-grown predefecating larva; cell 9, full-grown predefecating larva; cell 10, egg on firm provisions; cell 11, egg on soft provisions (hence, older than egg in cell 10). 26. Side view, dem¬ onstrating clusters of cells and varying contents of clusters I-V and contents of individual cells identified as follow: back-filled or containing dead immatures = grey; egg or live larvae = large stipples; live larvae = paral¬ lel bars. Upper back-filled lateral lost but possibly leading to back-filled cells (not shown); three descending back-filled tunnels at lowest part of nest presumably leading to unexplored old cells of previous generation(s). 27. Top view of same nest showing approximate distribution of cell clusters from above. 10 AMERICAN MUSEUM NOVITATES NO. 3893 ported by information presented in Sarzetti et al. (2014) concerning O. austera and is further discussed below concerning two of the larger nests of P. gloriosa that were studied. Cells are positioned with their long axes vertical (figs. 16-19) though a few were tipped as much as 10° from vertical. Cell lumens are about twice as long as their maximum diameters. Shapes presented in most diagrams are approximations; for the exact shape of an open cell, see figures 11 or 16. Table 1 presents nest statistics. Cell walls from all three localities were note¬ worthy because of their thickness and hardness, and because walls extended beyond the cells to cover the curved connecting lateral for perhaps 4 cm in front of the cell closure (figs. 4, 9, 18), thus providing an antechamber. Walls were of the same soil composition as the surround¬ ing soil and thus were not built with material from elsewhere. It was often possible when removing a cell from the ground to recover it as a hard nodule that was 5-6 cm long and 2 cm in width (figs. 3, 8, 14). Because of the irregular outer surface, the thickness of the wall varied from 2 to 3 mm or more, but gradually reduced in thickness toward the front end of the curved antechamber. This is clear evidence that the constructing female adds a hardening substance to the combined antechamber and cell wall that provides a strong barrier against predators or parasites. However, this substance does not control cell humidity during larval diapause since, when tested with a droplet of water on its inner surface, the antechamber wall immediately absorbed it, whereas the cell wall permits no absorption. Although the surface of the antecham¬ ber wall is smooth, it lacks the distinct but subdued luster of the cell wall, which is not only smooth but also moderately reflective. When the inner surface of the cell wall was tested with a drop of water, the droplet was still present 45 minutes later; even then the water had not penetrated the waterproof lining. That is, the female applies a hardening material to the cell wall as well as to the antechamber wall as well as a separate waterproofing coating only to the cell wall, both of unknown sources. The waterproofing material extends only to the cell entrance and not beyond, as was tested by applying a large water droplet to the area where the two surfaces met. The cell closure provided by the female after egg deposition is a circular disc with a dis¬ tinctly spiraled lower surface composed of four to five rows to the radius (fig. 12-15). The center of the disc is recessed about 2 mm compared with its periphery, and the entire disc tilts noticeably from a right angle with the long axis of cell. When the surface was tested with a water droplet, the droplet was immediately absorbed, thus suggesting that air exchange takes place through the closure, permitting the diapausing larva to survive months on end. However, another possible consequence of the permeability of the cell closure is that it may also permit water vapor to enter the cell, which might account for the rapid liquification of the stored provisions, as noted below, suggested by J. Cane (personal commun., 6/26/2017) with respect to another group of bees. An interesting discovery detected in cells from two of the localities was a series of trans¬ verse septa extending along the entrance tunnel from the cell closure toward the main burrow for at least 4 cm. Figure 10 shows six of the 11 septa that were revealed in one sample. The septa are composed of consolidated sandy material with the intervening spaces filled with loose sandy material. This suggests that, as the female backfills a tunnel, she repeatedly applies some